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Title:
Glyconanobiotics novel carbohydrated nanoparticle polymers
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English
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Abeylath, Thotaha Wijayahewage Sampath Chrysantha
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University of South Florida
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Tampa, Fla
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Subjects / Keywords:
Emulsion polymerization
Glyconanoparticle
Drug delivery
Antibiotic
Glycotargeting
Dissertations, Academic -- Chemistry -- Doctoral -- USF   ( lcsh )
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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Abstract:
ABSTRACT: Carbohydrates on the cell surface conjugates to proteins and lipids and participates in biological processes as glycoconjugates. Carbohydrate functionalized nanoparticles (glyconanoparticles) constitute a good bio-mimetic model of carbohydrate presentation at the cell surface and are currently centered on many glycobiological and biomedical applications. The most of the applications have been reported using gold glyconanoparticles. A brief review of gold glyconanoparticles and some of their applications will be discussed in Chapter I. Although metallic, semiconductor and magnetic glyconanoparticles have been reported, no polyacrylate glyconanoparticles have yet to be described. Chapter II describes the first preparation of carbohydrate functionalized polymer nanoparticles by microemulsion polymerization and their characterization using scanning electron microscopy, dynamic light scattering and 1H NMR spectroscopy. This methodology can generate a large number of furanose and pyranose nanoparticle derivatives with an average particle size of around 40 nm with the protected carbohydrate hydroxyl functionality as acetyl or dimethylacetal groups. Formation of larger glyconanoparticles of around 80 nm with 3-O-acryloyl-D-glucose and 5-O-acryloyl-1-methoxy-beta-D-ribofuranose reveals the influence of free hydroxyl groups in the monomer on the particle size during polymerization, a feature which is also apparently dependent on the amount of carbohydrate in the matrix. Preparation of glyconanoparticle antibiotics, or glyconanobiotics, by microemulsion of antibiotic-conjugated carbohydrate monomers demonstrates for the first time the use of glyconanoparticles as drug delivery vehicles in Chapter III. The conjugation of an acrylated hydrophobic carbohydrate moiety to the lipophilic antibiotic makes it even more lipophilic and suitable as a co-monomer in microemulsion polymerization with styrene/butyl acrylate. Novel carbohydrate-based acrylated acyl chlorides synthesized from glucose afford antibiotic monomers with enhanced lipophilicity in a one step procedure. These drug monomers and the corresponding glyconanobiotics prepared by conjugating antibiotics such as N-thiolated-beta-lactam, ciprofloxacin, and penicillin shows biological activity against S. aureus, MRSA and B. anthracis microbes. Glyconanoparticles prepared by microemulsion polymerization of 3-O-acryloyl-D-glucose and styrene/butyl acrylate may be potentially used as recognition units in carbohydrate ligand mediated targeted drug delivery. The binding capability of the surface-exposed carbohydrates on the nanoparticle can be detected by fluorescence spectroscopy utilizing pyranine and 4,4'-N,N-bis(benzyl-2-boronic acid)-bipyridinium dibromide as described in Chapter IV.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2007.
Bibliography:
Includes bibliographical references.
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by Thotaha Wijayahewage Sampath Chrysantha Abeylath.
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Title from PDF of title page.
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Document formatted into pages; contains 87 pages.
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Includes vita.

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oclc - 174144835
usfldc doi - E14-SFE0001948
usfldc handle - e14.1948
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ABSTRACT: Carbohydrates on the cell surface conjugates to proteins and lipids and participates in biological processes as glycoconjugates. Carbohydrate functionalized nanoparticles (glyconanoparticles) constitute a good bio-mimetic model of carbohydrate presentation at the cell surface and are currently centered on many glycobiological and biomedical applications. The most of the applications have been reported using gold glyconanoparticles. A brief review of gold glyconanoparticles and some of their applications will be discussed in Chapter I. Although metallic, semiconductor and magnetic glyconanoparticles have been reported, no polyacrylate glyconanoparticles have yet to be described. Chapter II describes the first preparation of carbohydrate functionalized polymer nanoparticles by microemulsion polymerization and their characterization using scanning electron microscopy, dynamic light scattering and 1H NMR spectroscopy. This methodology can generate a large number of furanose and pyranose nanoparticle derivatives with an average particle size of around 40 nm with the protected carbohydrate hydroxyl functionality as acetyl or dimethylacetal groups. Formation of larger glyconanoparticles of around 80 nm with 3-O-acryloyl-D-glucose and 5-O-acryloyl-1-methoxy-beta-D-ribofuranose reveals the influence of free hydroxyl groups in the monomer on the particle size during polymerization, a feature which is also apparently dependent on the amount of carbohydrate in the matrix. Preparation of glyconanoparticle antibiotics, or glyconanobiotics, by microemulsion of antibiotic-conjugated carbohydrate monomers demonstrates for the first time the use of glyconanoparticles as drug delivery vehicles in Chapter III. The conjugation of an acrylated hydrophobic carbohydrate moiety to the lipophilic antibiotic makes it even more lipophilic and suitable as a co-monomer in microemulsion polymerization with styrene/butyl acrylate. Novel carbohydrate-based acrylated acyl chlorides synthesized from glucose afford antibiotic monomers with enhanced lipophilicity in a one step procedure. These drug monomers and the corresponding glyconanobiotics prepared by conjugating antibiotics such as N-thiolated-beta-lactam, ciprofloxacin, and penicillin shows biological activity against S. aureus, MRSA and B. anthracis microbes. Glyconanoparticles prepared by microemulsion polymerization of 3-O-acryloyl-D-glucose and styrene/butyl acrylate may be potentially used as recognition units in carbohydrate ligand mediated targeted drug delivery. The binding capability of the surface-exposed carbohydrates on the nanoparticle can be detected by fluorescence spectroscopy utilizing pyranine and 4,4'-N,N-bis(benzyl-2-boronic acid)-bipyridinium dibromide as described in Chapter IV.
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Glyconanobiotics: Novel Carbohydrated Nanoparticle Polymers by Thotaha Wijayahewage Sampath Chrysantha Abeylath A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemistry College of Arts and Sciences University of South Florida Major Professor: Edward Turos, PhD. Julie P. Harmon, PhD. Kirpal S. Bisht, PhD. Roman Manetsch, PhD. Date of Approval: April 06, 2007 Keywords: Emulsion polymerization, Glyconanoparticle, Drug delivery, Antibiotic, Glycotargeting Copyright 2007, Thotaha Wijayahewage Sampath Chrysantha Abeylath

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ACKNOWLEDGMENTS I would like to thank many people for their assistance and guidance for the completion of this research in the Department of Chemistry at the University of So uth Florida. First and foremost, I am grateful to my research advisor, Professor Edward Turos, who has taught me the art of organic chemistry and the value of carbohydrate research. Throughout the years, Professo r Turos has maintained an environment rich in intellectual thought for which to mature in as a scientist. I am also grateful to him for granting me the freedom to conduct investigations that were of interest. The phe nomenal experience of working in Dr. Turos’ laboratory will serve me well in the future endeavors for which I will always be indebted. I was obliged to receive direction from an outsta nding committee during my studies at the University of South Florida. I am grateful to Professor Julie P. Harmon, Professor Kirpal Bisht and Professor Roaman Manetsch for their valuable advices which made my research experience an enjoyable one. I am grateful to all of my cu rrent and former colleag ues for their assistance and support. First and foremost, my former lab mates whose support was inva luable for me as a new graduate student: Bart A. Heldreth, Timothy E. Long, Cristina M. Coates and Jeung-Yeap Shim. Also I would like to thank Yang Wang and J. Michelle Leslie who joined the group at the same time. Finally, I would like to thank other remarkable individuals who joined the lab: Dr. Seyoung Jang, Julio Garay, Praveen Ramaraju, Kerriann Greenhalgh, Marci Culbreath, Kevin Revell, Dr. Tyler Schertz, Dr. Ivan Perez, Dr. Suresh Reddy and Dr. Rajesh Mishra. It was a wonderful and pleasure experience to work with them over the years. I would like to thank Dr. Randy W. Larsen of Department of Chemistry for assisting me in fluorescence studies in his lab and Sonja Dickey of the Department of Biology for her efforts made in the biological studies. Finally, I thank my parents, Dayasena Abeylath and Yolie Cooray. They provided everything needed for me to succeed in life. Also I want to thank my wife, Ruchira, and my son, Ranuk, for their love and support. I would extend my thanks and love to all of my family members in Sri Lanka includi ng my brother, Asiri, and mother-in-law.

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TABLE OF CONTENTS LIST OF TABLES iv LIST OF FIGURES v LIST OF SCHEMES vii LIST OF SPECTRA ix LIST OF ABBREVIATIONS xi ABSTRACT xii CHAPTER I GLYCONANOPARTICLES IN GLYCOSCIENCE AND BIOMEDICINE 1 1.1 Introduction 1 1.2 Gold glyconanoparticles 2 1.3 Glyconanoparticles to study protein-protein interactions 4 1.4 Glyconanoparticles as biolabels 5 1.5 Glyconanoparticles in biomedicine 6 1.6 Conclusions 7 CHAPTER II GLYCOSYLATED POLYACRYLATE NANOPARTICLES BY EMULSION POLYMERIZATION 8 2.1 Introduction 8 2.2 Polymerization in microemulsion 9 2.3 Synthesis of carbohydrate acrylate monomers 11 2.3.1 O -isopropylidene protecte d carbohydrate acrylates 1 4 12 2.3.2 O -acetal protected acrylamide fr om glucosamine 13 2.4 Glyconanoparticle preparation by microemulsion polymerization 14 2.5 Glyconanoparticle characterization 15 2.5.1 Scanning Electron Microscopy (SEM) analysis 16 2.5.2 Dynamic Light Scattering (DLS) analysis 17 2.5.3 1H NMR analysis 18 i

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2.6 Influence of bulky aryl protecting groups in the carbohydrate acrylate monomer on glyconanoparticle preparation. 19 2.6.1 1O -Acryloyl-2,3,5-triO -benzyl-D-ribofuranose ( 13 ) 19 2.6.2 N -acryloyl-1,3,4,6-tetraO -benzoyl-D-glucosamine ( 14 ) 20 2.7 Influence of free hydroxyl groups of the monosaccharide acrylate monomer on the formation of glyconanoparticles. 21 2.8 Nucleoside-bearing polyacrylate nanoparticles 23 2.8.1 4N -acetyl-5’O -acryloyl-2’,3O -isopropylidenecytidine 23 2.9 Conclusions 24 CHAPTER III GLYCONANOBIOTICS: SYNTHESIS AND BIOLOGICAL ACTIVITY 25 3.1 Introduction 25 3.2 Synthesis of novel carbohydrate-based acyl chlorides 26 3.2.1 3O -Acryloyl-1,2O -isopropylidene-5,6 bis((chlorosuccinyl)oxy)-D-glucofuranose 27 3.2.2 6O -Acetyl-3O -Acryloyl-1,2O -isopropylidene-5-((chlorosuccinyl)oxy)-DGlucofuranose 28 3.3 Synthesis of novel antibiotic-conjugated carbohydrate monomers 28 3.3.1 N -Thiolated -lactam-conjugated glucose acrylate 28 3.3.2 Ciprofloxacin-conjugated glucose acrylate 31 3.3.3 Penicillin-conjugated glucose acrylate 34 3.4 Glyconanobiotics by emulsion polymerization 36 3.5 Biological activity 37 3.5.1 Biological activity of monomers against S. aureus MRSA and B. anthracis 38 3.5.2 Biological activity of glyconanobiotics against S. aureus MRSA and B. anthracis 38 3.6 Conclusions 39 CHAPTER IV PREPARATION OF POLYACRYLATE GLYCONANOPARTICLES WITH SURFACE CARBOHYDRATES 40 4.1 Introduction 40 4.2 Preparation of acrylated glucose monomers 41 4.3 Microemulsion polymerization of acrylated glucose monomers 42 4.4 Detection of the binding ability of glyconanoparticles 44 4.4.1 Sugar detection by boronic acids 44 4.4.1.1 4’,4’-N’,N’-bis(benzyl-2-boronic acid)-bipyridinium dibromide (o-BBV) 44 4.4.2 Binding of o-BBV to 3-acryloyl-D-glucose 47 4.4.3 Binding of o-BBV to glyconanoparticles 49 4.5 Conclusions and future work 52 ii

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CHAPTER V MATERIALS AND METHODS 53 1H AND 13C SPECTRA 62 REFERENCES 84 ABOUT THE AUTHOR End Page iii

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LIST OF TABLES Table 2.1: Formulation used for the emulsion polymerization 15 Table 2.2: Average particle sizes of glyconanoparticles GNP 1-5 17 Table 3.1: Formulation used for the emulsion polymerization to prepare antibiotic-conjugated glyconanoparticles GNB 34 GNB 37 and GNB 40 37 Table 3.2: Average particle sizes of glyconanoparticles GNP 34, GNP37 and GNP40 as determined by dynamic light scattering 37 Table 3.3: MIC values for antibiotic-conjugated carbohydrate monomers 38 Table 3.4: MIC values for glyconanobiotics 38 Table 4.1: Formulation used for the emulsion polymerization to prepare glyconanoparticle with surface carbohydrates 43 Table 4.2: Average particle sizes of glyconanoparticles 44 iv

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LIST OF FIGURES Figure 1.1 : Structures of GM3, Gg3 and KDNGM3 glycosphingolipids 1 Figure 1.2: Structures of thiol-derivatized neoglycoconjugates; monosaccharide (glucose), disaccharide (maltose) and tetrasaccharide (Ley) 4 Figure 1.3: A schematic illustration for the colorimetric detection of protein-protein interactions 5 Figure 1.4: Mannose-encapsulated gold nanoparticles binding to the specific receptor of type 1 pili 6 Figure 1.5: Possible mechanism of lactose glyconanoparticles in anti-adhesive therapy 7 Figure 2.1: Chemical structures of glycopolymers prepared with acetonide protected (a) glucose and (b) galactose 8 Figure 2.2: Schematic representation of the micelle nucleation model 10 Figure 2.3: Carbohydrate monomers used for the formation of glyconanoparticles 11 Figure 2.4: A vial containing a representa tive carbohydrate-bearing polyacrylate emulsion 14 Figure 2.5: SEM image of glyconanoparticles prepared from 3O -acryloyl-1,2:5,6-diO isopropylidene-D-glucofuranose ( 1 ) and ethyl acrylate by emulsion polymerization 16 Figure 2.6: Particle size distribution of glyconanoparticles prepared from 3O -acryloyl-1,2:5,6-diO isopropylidene-D-glucofuranose ( 1 ) and ethyl acrylate by emulsion poly merization 17 Figure 2.7: Typical 1H NMR spectra of (a) 3O -acryloyl-1,2:5,6-diO -isopropylidine -D-glucofuranose ( 1 ) and (b) the glycopolymer obtained from the emulsion after the film formation. 18 Figure 2.8: 1O -Acryloyl-2,3,5-triO -benzyl-D-ribofuranose ( 13 ) and 2N -acryloyl -1,3,4,6-tetraO -benzoyl-D-glucosamine ( 14 ). 19 Figure 2.9: Comparison of the effects of using an O -protected versus O -unprotected monosaccharide acrylate on nanoparticle size 22 Figure 3.1: Structures of acrylated antibiotic Monomers of N -thiolated -lactam ( 24 ), penicillin ( 25 ) and ciprofloxacin ( 26 ) 25 Figure 3.2: Structures of diacyl chloride 27 and acyl chloride 28 26 Figure 3.3: 1H NMR spectrum of N-sec -butylthio -lactam monomer 34 30 Figure 3.4: ESI-MS spectrum of N-sec -butylthio -lactam monomer 34 30 v

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Figure 3.5: 1H NMR spectrum of mono-ciprofloxacin monomer 37 33 Figure 3.6: ESI-MS spectrum of mono-ciprofloxacin monomer 37 33 Figure 4.1: A cartoon showing the use of carbohydrate ligands to target protein receptors in targeted drug delivery to bacterial cell 40 Figure 4.2: Glucose-functionalized polyacrylate nanoparticles with (a) short and (b) long tether lengths 41 Figure 4.3: Acrylated D-glucose monomers 41 Figure 4.4: Pyranine ( 47 ) and o-BBV ( 48 ) 45 Figure 4.5: Fluorescence spectra of pyranine 48 Figure 4.6: Fluorescence spectra of pyranine 50 Figure 4.7: Dibenzofuran-4-boronic acid 51 Figure 4.8: Post-functionalization of glyconanoparticles 52 vi

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LIST OF SCHEMES Scheme 1.1: Preparation of gold glyconanoparticles 3 Scheme 1.2: Synthesis of mannose-encapsulated gold nanoparticles from thiomannosyl dimer 6 Scheme 2.1: Synthesis of O -isopropylidene protected carbohydrate acrylates 12 Scheme 2.2: Synthesis of N -acryloyl 1,3,4,6-tetraO -acetyl-D-glucosamine 13 Scheme 2.3: Preparation of glyconanoparticles by emulsion polymerization 14 Scheme 2.4: Synthesis of 2,3,5-triO -benzyl-D-ribofuranose-1-acrylate 19 Scheme 2.5: Synthesis of N -acryloyl-1,3,4,6-tetraO -benzoyl-D-glucosamine 20 Scheme 2.6: Deacetonation of carbo hydrate acrylates 1 and 5 21 Scheme 2.7: Synthesis of 4N -acetyl-5’O -acryloyl-2’,3O -isopropylidenecytidine 23 Scheme 3.1: Synthesis of 3O -acryloyl-1,2O -isopropylidene-5,6 bis((chlorosuccinyl)oxy)-D-glucofuranose 27 Scheme 3.2: Synthesis of 6O -acetyl-3O -acryloyl-1,2O -isopropylidene -5-((chlorosuccinyl)oxy)-D-glucofuranose 28 Scheme 3.3: Synthesis of N-sec -butylthio -lactam monomer 34 29 Scheme 3.4: Synthesis of bis-ciprofloxacin monomer 36 31 Scheme 3.5: Synthesis of mono-ciprofloxacin monomer 37 32 Scheme 3.6: Synthesis of penicillin-c ontaining acrylate monomer 40 35 Scheme 3.7: Preparation of glyconanobiotics by emulsion polymerization 36 Scheme 4.1: Synthesis of monomer 41 42 Scheme 4.2: Preparation of glyconanoparticles with surface carbohydrates by emulsion polymerization 43 Scheme 4.3: Equilibrium established between the aryl boronic acid and a carbohydrate; and its dependency on pH 45 vii

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Scheme 4.4: Synthesis of 4’,4’-N’,N’-bis(benzyl-2-boronic acid)-bipyridinium dibromide (o-BBV) 46 Scheme 4.5: Reversible complexation reaction of o-BBV/3-acryloyl-D-glucose 47 Scheme 4.6: Binding of glyconanoparticles with o-BBV 49 viii

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LIST OF SPECTRA Spectrum 6.1: 1H NMR (250 MHz, CDCl3) ( 1 ) 62 Spectrum 6.2: 13C NMR (63 MHz, CDCl3) ( 1 ) 62 Spectrum 6.3: 1H NMR (250 MHz, CDCl3) ( 2 ) 63 Spectrum 6.4: 13C NMR (63 MHz, CDCl3) ( 2 ) 63 Spectrum 6.5: 1H NMR (250 MHz, CDCl3) ( 3 ) 64 Spectrum 6.6: 13C NMR (63 MHz, CDCl3) ( 3 ) 64 Spectrum 6.7: 1H NMR (250 MHz, CDCl3) ( 4 ) 65 Spectrum 6.8: 13C NMR (63 MHz, CDCl3) ( 4 ) 65 Spectrum 6.9: 1H NMR (250 MHz, CDCl3) ( 5 ) 66 Spectrum 6.10: 13C NMR (63 MHz, CDCl3) ( 5 ) 66 Spectrum 6.11: 1H NMR (250 MHz, CDCl3) ( 13 ) 67 Spectrum 6.12: 13C NMR (63 MHz, CDCl3) ( 13 ) 67 Spectrum 6.13: 1H NMR (250 MHz, CDCl3) ( 14 ) 68 Spectrum 6.14: 1H NMR (400 MHz, D2O) ( 19 ) 69 Spectrum 6.15: 13C NMR (63 MHz, D2O) ( 19 ) 69 Spectrum 6.16: 1H NMR (400 MHz, D2O) ( 20 ) 70 Spectrum 6.17: 13C NMR (100 MHz, D2O) ( 20 ) 70 Spectrum 6.18: 1H NMR (400 MHz, CDCl3) ( 23 ) 71 Spectrum 6.19: 13C NMR (63 MHz, CDCl3) ( 23 ) 71 Spectrum 6.20: 1H NMR (400 MHz, CDCl3) ( 27 ) 72 Spectrum 6.21: 13C NMR (100 MHz, CDCl3) ( 27 ) 72 Spectrum 6.22: 1H NMR (400 MHz, CDCl3) ( 28 ) 73 ix

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Spectrum 6.23: 13C NMR (100 MHz, CDCl3) ( 28 ) 73 Spectrum 6.24: 1H NMR (400 MHz, CDCl3) ( 30 ) 74 Spectrum 6.25: 13C NMR (100 MHz, CDCl3) ( 30 ) 74 Spectrum 6.26: 1H NMR (400 MHz, CDCl3) ( 32 ) 75 Spectrum 6.27: 13C NMR (100 MHz, CDCl3) ( 32 ) 75 Spectrum 6.28: 13C NMR (100 MHz, CDCl3) ( 34 ) 76 Spectrum 6.29: 1H NMR (400 MHz, CDCl3) ( 36 ) 77 Spectrum 6.30: 13C NMR (100 MHz, CDCl3) ( 37 ) 78 Spectrum 6.31: 1H NMR (400 MHz, CDCl3) ( 40 ) 79 Spectrum 6.32: 1H NMR (400 MHz, CDCl3) ( 41 ) 80 Spectrum 6.33: 1H NMR (400 MHz, CDCl3) ( 45 ) 81 Spectrum 6.34: 13C NMR (63 MHz, CDCl3) ( 45 ) 81 Spectrum 6.35: 1H NMR (400 MHz, CDCl3) ( 46 ) 82 Spectrum 6.36: 13C NMR (63 MHz, CDCl3) ( 46 ) 82 Spectrum 6.37: 1H NMR (250 MHz, D2O) ( 48 ) 83 x

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LIST OF ABBREVIATIONS alpha Ar aryl Ac acetyl beta Bn benzyl br broad (spectral) Bu butyl Bz benzoyl oC degree Celsius 13C carbon-13 CH2Cl2 dichloromethane DLS dynamic light scattering DMAP 4-dimethylaminopyridine DMSO dimethylsulfoxide EDCI 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide ESI electrospray ionisation Et ethyl Et3N triethylamine EtOAc ethyl acetate g gram(s) 1H proton hr hour(s) J coupling constant(s) Me methyl MeOH methanol mg milligram(s) min. minutes mmol millimole(s) mol mole(s) MRSA methicillin-resistant Staphylococcus aureus MS mass spectroscopy MWCO molecular weight cut-off NBS N-bromosuccinimide ppm parts per million Ph phenyl Py pyridine RT room temperature SEM scanning electron microscopy TFA trifluoroacetic acid Ts p-toluenesulfonyl xi

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Glyconanobiotics: Novel Carbohydrated Nanoparticle Polymers Thotaha Wijayahewage Sampath Chrysantha Abeylath ABSTRACT Carbohydrates on the cell surface co njugates to proteins and lipids and participates in biological processes as glycoconjugates. Carbohydrate functiona lized nanoparticles (glyconanoparticles) constitute a good bio-mimetic model of carbohydrate presentatio n at the cell surface and are currently centered on many glycobiological and biomedical applications. The most of the applications have been reported using gold glyconanoparticles. A brief review of gold glycon anoparticles and some of their applications will be discussed in Chapter I. Although metallic, semiconductor and magnetic glyconanoparticles have been reported, no polyacrylate glyconanoparticles have yet to be described. Chapter II describes the first preparation of carbohydrate functionalized polymer nanoparticles by microemulsion polymerization and their characterization using scanning electron microscopy, dynamic light scattering and 1H NMR spectroscopy. This methodology can generate a large number of furanose and pyranose nanoparticle derivatives with an average particle size of around 40 nm with the protected carbohydrate hydroxyl f unctionality as acetyl or dimethylacet al groups. Formation of larger glyconanoparticles of around 80 nm with 3O -acryloyl-D-glucose and 5O -acryloyl-1-methoxy-Dribofuranose reveals the influence of free hydroxyl groups in the monomer on the particle size during polymerization, a feature which is also apparently dependent on the amount of carbohydrate in the matrix. Preparation of glyconan oparticle antibiotics, or glyconanobiotics by microemulsion of antibiotic-conjugated carbohydrate monomers demonstrates for the first time the us e of glyconanoparticles as drug delivery vehicles in Chapter III. The conjug ation of an acrylated hydro phobic carbohydrate moiety to the lipophilic antib iotic makes it even more lipophilic and su itable as a co-monomer in microemulsion polymerization with styrene/butyl acr ylate. Novel carbohydrat e-based acrylated acyl chlorides synthesized from glucose afford antibiotic monomers with enhanc ed lipophilicity in a one st ep procedure. These drug monomers and the corresponding glyconanobiotics prepared by conjugating antibiotics such as N -thiolated-lactam, ciprofloxacin, and penicillin shows biological activity against S. aureus MRSA and B. anthracis microbes. Glyconanoparticles prepared by microemulsion polymerization of 3O -acryloyl-D-glucose and styrene/butyl acrylate may be potentially used as recognition units in carbohydrate ligand mediated targeted drug delivery. The binding capability of the surface-exp osed carbohydrates on the nanoparticle can be detected by fluorescence spectroscopy utilizing pyranine and 4,4’N N -bis(benzyl-2-boronic acid)bipyridinium dibromide as described in Chapter IV. xii

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CHAPTER I GLYCONANOPARTICLES IN GLYCOSCIENCE AND BIOMEDICINE 1.1 Introduction Carbohydrates are, together with nucleic acids an d proteins, important molecules of life. Much is already known about the stru cture, interactions and function of nucleic acids and proteins. However, the role of carbohydrates in th e cell is less clear. The surf ace of mammalian cells is co vered by a de nse coating of carbohydrates named glycocalyx.1 In the glycocalyx, carboh ydrates appear mainly conjugated to proteins and lipids (glycoproteins and glycolipids) and it is as glycoconjugates that they develop their biological function. Now, it is known that these complex oligosaccharides are involved in the control of many normal and pathological processes. 2,3 Hakomori in 1989 proposed that the first phase of cell adhesion initiates through a carbohydrate-carbohydrate interaction be tween multivalent glycosphingolipid-domains. This initial step is followed by protein-pr otein interactions between adhesion receptors and pr otein-carbohydrate interactions between lectins and glycoconjugates. Embryogenesis, metastasis and other cellular proliferation processes are mediat ed by carbohydrate-carbohydrate in teractions, according to Hakomori. 4-9 Figure 1.1 : Structures of GM3, Gg3 and KDNGM3 glycosphingolipids O O O O O C13H27 OH HO OH OH AcHN HO HOOC HO OH OH OH NHCOC17H35 OH O HO GM3O O HO O C13H27 OH O OH OH OH NHCOC17H35 OH O HO O OH OH HO NHAc Gg3O O O O O C13H27 OH HO OH OH HO HO HOOC HO OH OH OH NHCOC17H35 OH O HO (KDN)GM3 1

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Several glycosphingolipids involved in carbohydrate-carbohydrate interactions have already been identified. For example, the interaction between the glycosphingolipids GM3 and Gg3 has been proposed to be involved in metastasis of melanoma cells in mouse. 6-8 It has been demonstrated recen tly that the interaction between KDNGM3 and Gg3 is involved in the binding of sper m to egg membrane in rainbow trout fertilization 9(Figure 1.1). Since nanoparticles and biomolecules are of similar dimensions, nanoparticles can imitate biomolecular presentation in cellular systems, probing the mechanisms of biological processes, as well as developing chemical means for handling and manipulating biological components.10 Although several groups have prepared nanoparticles functionalized with proteins and DNA11 during the last 10 years for this purpose, carbohydrates had not been used to functionalize nan oparticles until the first synthesis of carbohydrate functionalized gold nanoparticles (glyconanoparticles) were reported in 2001.12 The glyconanoparticles can mimic carbohydrate presentation to glycoproteins or glycosphingolipids patches, by providing a glycocalyxlike surface, presenting carbohydrates in a globular and polyvalent co nfiguration on their surface. Also glyconanoparticles have unusual physical properties due to the quantum size effect that can be used for the detection and evaluation of the interac tions where carbohydrates are involved. 13,14 In spite of the short history of these new glyconanomaterials, some applications have already been reported in the field of chemical glycobiology mainly focusing on the study and evaluation of carbohydrate interactions. Furthermore, reports have appeared on glyconanoparticles in applications to biomedicine. Gold and silver glyconanoparticles, semiconductor glycol-quantum dots and magnetic glyconanoparticles are the three different types of nanoparticles functionalized with carbohydrates so far and the most of the applications have been reported using gold glyconanoparticles. This chapter discusses the preparation of gold glyconanoparticles and some of their applications in glycoscience and biomedicine. 1.2 Gold glyconanoparticles Gold nanoparticles are the most stable metal nanoparticles. The first synthesis of gold glyconanoparticles was carried out by adding a methanolic solution of thiol-derivatized carbohydrate compounds called neoglycoconjugates to an aqueous solution of tetrachloroauric acid (HAuCl4) and subsequent reduction with NaBH4.12 Three neoglycoconjugates of two biologically significant oligosaccharides, the disaccharide lactose (Gal (1-4)Glc 1-OR) and the trisaccharide Lex (Gal (1-4)[Fuc (1-3)]GlcNAc 1-OR), were used for this purpose (Scheme 1.1). 2

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Scheme 1.1: Preparation of gold glyconanoparticles O HO OH O OH OH O OH HO OH O X S 3 2O O NHAc OH O X S 3O O OH OH HO OH O OH OH OH H3C 2 X=O,CH2X=CH2O O S 2 Au O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O disaccharidetetrasaccharide HAuCl4NaBH4MeOH This approach opened the way to tailoring glyconanopartic les with a variety of carbohydrate ligands. Following this method, the synthesis of different glyconanoparticles functionalized with the monosaccharide glucose, disaccharide maltose (Glc 1-4Glc 1)15 and tetrasaccharide Ley ([Fuc 1-2]Gal 1-4[Fuc 1-2]GlcNAc 1) 16 have been reported (Figure 1.2). 3

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Figure 1.2: Structures of thiol-derivatized neoglycoconjugates; monosaccharide (glucose), disaccharide (maltose) and tetrasaccharide (Ley) HO O OH HO OH O O S 3 2O HO OH OH 3O 2O O NHAc OH O O OH OH OH H3C O OH HO HO OH O S O O OH OH HO O O OH OH H3C OH 3 S 2 monosaccharide disaccharide tetrasaccharide Different linkers of hydrophobic (alkyl) and hydrophilic (p olyethylene glycol derivatives) have been used to bind the carbohydrate to the gold core. 15 The glyconanoparticles prepared in this way are water soluble, stable in solution for years, non-cytotoxic and exceptionally small (less than 2 nm). 13,17 1.3 Glyconanoparticles to study protein-protein interactions Gold glyconanoparticles have been used extensively to study protein-carbohydrate interactions. 18 Also more recently, Chen et al. have applie d the visible color change for the det ection of protein-pr otein interactions 19 using glyconanoparticles. Thyroglobulin, a binding protein for glycoproteinConcanavalin A, was added to mannopyranoside-encapsulated glyconanoparticles complexed to Concanavalin A. The color change from purple to pinkish red, due to the dispersion of glyc onanoparticles, allowed the qualitatively and quantitatively evaluation of the thyroglobulin-Concanavalin A interaction (Figure 1.3). 4

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Figure 1.3: A schematic illustration for the colorimetric detection of protein-protein interactions. (a) gold glyconanoparticles (b) agglomeration of the glycoparticles via glyconanoparticle-ConA interactions (purple) (c) dissipation of the glyconanoparticles by formation of thyroglobulin-ConA interactions (pinkish red). : Concanavalin A :Thyroglobulin : Glyconanoparticles (a) (b) (c) 1.4 Glyconanoparticles as biolabels Glyconanoparticles can be used as efficient labeling probes in a biological system. Lin et al. have demonstrated the selective binding of mannose-encapsulat ed gold nanoparticles to adhesin FimH of type 1 pili in Escherichia coli by transmission electron microscopy (TEM). 20 Adherence is often an essential step in bacterial pathogenesis. To effectively adhere to host su rfaces, many bacteria produce multiple adherence factors called adhesins. Type 1 pili are fila mentous protein st ructures that extend from the surface of many gramnegative organisms and are composed of FimA, FimF, FimG, and FimH proteins. 21 FimA accounts for more than 98% of the protein and FimH is uniquely responsible for the binding to D-mannose. 22 They have used two E. coli strains ORN178 and ORN208 in experiments to confirm the binding of mannose nanoparticles, synthesized from thiomannosyl dimmer (Scheme 1.2), to FimH. The ORN178 strain expresses wild-type type-1 pili, whereas the ORN208 strain is deficient of the FimH and expresses abnormal type 1 pili that fail to mediate D-mannose specific binding. After incubating the two bacterial strains separately with mannose gold nanoparticles and examining by TEM, selective binding of the glconanoparticles to the ORN178 has been observed (Figure 1.4). The selective binding of these gl yconanoparticles to bacterial type I pili presented a novel method of labeling specific r eceptors for carbohydrates on the cell surface using carbohydrate-conjugated nanoparticles. 5

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Scheme 1.2: Synthesis of mannose-conjugated gold nanoparticles from thiomannosyl dimer O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O Au O O HO HO S HO OH 2 HAuCl4NaBH4 Figure 1.4: Mannose-conjugated gold nanoparticles bindin g to the specific recep tor of type 1 pili O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O Au ORN178 ORN208 Selective binding 1.5 Glyconanoparticles in biomedicine Since glyconanoparticles represent good biomimetic models of carbohydrate-mediated biological processes, they have been applied in a variety of biom edical applications. Lactose gold glyconanoparticles have been shown as potential tools in anti-adhesive therapy.23 Metastasis is the origin of the poor prognosis of most cancers. One of the critical st eps in metastasis is the adhesion of tumor cells to the vascular endothelium. After adhesion, tumor cells migrate and create new tumor foci. 24 Interactions between tum our-associated antigens and epithelial cell selectins promote tumor cell metastasis. 25 In addition to this mechanism, carbohydratecarbohydrate interactions between gl ycosphingolipids expressed on the tu mor and endothelial cell surfaces also seem to be involved in the critical adhesion step. 26 A carbohydrate-carbohydrate interaction between GM3 expressed in a murine melanoma cell line (B16) and Gg3 of endothelium cells has been proposed to be involved in the first adhesion step of tumor cells to endothelium before transmigration. 27,28 Therefore, inhibition of this step by glyconanoparticles that pres ent carbohydrate antigens expressed eith er in the tumor or the endothelium cells might provide effective anti-adhesion therapy as shown in Figure 1.5. 6

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Figure 1.5: Possible mechanism of lactose glycona noparticles in anti-adhesive therapy23 Tumoral cells Endothelial cells Glycoconjugates Glyconanoparticles Cellular adhesion via carbohydratecarbohydrate interactions Anti-adhesion therapy using glyconanoparticles Tumoral cells transmigration through endothelial cells Invasion and metastasis Tumor Based on the involvement of the antigen lactos ylceramide in cell adhesion, lactose-conjugated glyconanoparticles have been tested as a potential inhibitor of the binding of melanoma cells to endothelium.23 According to the experiment designed for the evaluation of the anti-metastasis potential of the glyconanoparticles, mice were injected with mela noma cells pre-incubated with lactose-bound gold nanoparticles. After three weeks, the animals were s acrificed and both lungs were evaluated under the microscope for analysis of tumor foci. 70% of the tumor growth was found to be inhibited, as compared with the group inoculated only with melanoma cells. 1.6 Conclusions In this chapter, the preparation of water-soluble and stable gold glyconanoparticles and some of their applications were discussed. These nanomaterials are good biomimetic models of carbohydrate presentation at the cell surface. These multivalent systems, having ca rbohydrate-mediated interactions, have opened up applications in the field of glycobiology. Also the simple and flexible preparation of these glyconanoparticles and their interesting physical, chemical and biological properties allow them to find utility in biomedicine. The use of carbohydrate ligands to target protein receptors, a process termed glycotargeting, is a promising approach in targeted drug delivery. Drug-conjugated glyconanoparticles provide a variety of new avenues and opportunities in ligand-mediated targeted drug delivery. In the next chapter, the preparation of polyacrylate glyconanoparticles by emulsion polymerization will be discussed for the biomedical uses of antibiotic-laden glyconanoparticles. 7

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CHAPTER II GLYCOSYLATED POLYACRYLATE NANOPARTIC LES BY EMULSION POLYMERIZATION 2.1 Introduction Synthetic carbohydrate polymers, with biocompatible and biodegradable properties, are increasingly used for investigating glycopolymer-protein interactions.29 Also these glycopolymers have been widely investigated for pharmaceutical a nd medical applications in the tr eatment of infectious diseases.30 Several types of glycopolymers (as shown in Fig. 2.1) have been synthesized by free radical polymerization of vinyl monomers with pendent saccharide residues 31 and carbohydrate-ca rrying acrylates. 32, 33 Glyconanoparticles have been used in a variety of applications as mentioned in the previous chapter and all of these nanomaterials have been prepared by conjugating carbohydrates to metallic or semiconducting nanostructures. Although the preparation of poly( -benzyl L-glutamate) (PBLG) or poly(lactic acid) (PLA) nanoparticles using a carbohydrate-carrying polystyr ene (PS) amphiphilic polymer, which serves as both an emulsifier and a surface coating, has been reported as a liver-specific targeting material to deliver drugs34,35, no polyacrylate glyconanoparticles have yet to be de scribed. Recently, the Turos group repo rted the formation of antibacterially Figure 2.1: Chemical structures of glycopolymers prepared with protected (a) glucose and (b) galactose. O O HC O H2C nO O O O O O O O O O O C H2C n CH3 (a) (b) -active polyacrylate nanoparticles by emulsion polymerization using N -thiolated -lactam acrylates as comonomers in a soluble mixture of butyl acrylate-styrene.36 These antibiotic-attached polyacrylate nanoparticles showed activity against Staphylococcus aureus ( S. aureus ) and methicillin-resistant Staphylococcus aureus (MRSA). This development sparked the idea of carbo hydrate-bearing polyacrylat e nanoparticles (polymer glyconanoparticles) by microemulsion polymerization serving as new drug delivery vehicles. The carbohydrate itself can thereby serve as a linker unit and as a recognition unit in carbohydrate-ligand mediated targeted drug delivery (glycotargting) systems. This chapter will pres ent the preparation and char acterization of the polymer glyconanoparticles and investigation of the effect of chemical structure of the carbohydrate monomer on the glyconanoparticle preparation. 8

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2.2 Polymerization in microemulsion The first description of a microemulsion was published by Hoar and Schulman in 1943.37 Sixteen years later, Schulman et al. also postulated a mechan ism for its formation and introduced the notation “ microemulsion ”. 38 Polymerization in a microemulsion is a polymerization technique which allows the synthesis of ultrafine latex particles in the nanomete r range and with narrow size distribution. They are isotropic, transparent or translucen t, and thermodyna mically stable. A typical microemulsion polymerization formation comprises monomer, water, surfactant and a water soluble initiator. Surfactant (or the emulsifier) refers to a surface active agent and contains both hydrophobic and hydrophilic components. These molecules are semi-soluble in both organic and aqueous solvents accumulating at the interface and changing the surface tensi on. Based on their dissociation in water, surfactants are classified as anionic, nonionic, cationic, zwitterionic and nonionic su rfactants. The initia tor reacts with a monomer to form an intermediate compound capable of linking successively with a large number of other monomers into a polymeric compound. In a free radical polymerization, th e choice of polymerization initiator depends mainly on two factors: its solubility and its decomposition temperature. If the polymerization is performed in water, the initiator should be water so luble and the decomposition temperature of the initiator must be at or below the boiling point of water. The reaction system is characterized by the emulsifi ed monomer droplets dispersed in the continuous aqueous phase with the aid of an o il-in-water surfactant at the very be ginning of polymerization. Monomer swollen micelles exist in the reaction system provided th at the concentration of surfactant in the aqueous phase is above its critical micelle concentration (CMC). Only a small fraction of the relatively hydrophobic monomer is present in the micelles or dissolved in the aqueous phase. Most of the monomer molecules dwell in the giant monomer reservoirs (monomer droplets). The polymerizat ion is initiated by the add ition of initiator. According to the micelle nucleation model, waterborne free radicals first polymerize with monomer molecules dissolved in the continuous aqueous phase. This would result in the in creased hydrophobicity of oligomeric radicals. When the critical chain length is achieved, these oligomeric radicals become so hydrophobic that they show a strong tendency to enter the monomer-swollen micelles and then continue to propagate by reacting with those monomer molecules therein. As a consequence, mono mer-swollen micelles are succes sfully transformed into particle nuclei. These embryo particles continue to grow by acquiring the r eactant species from monomer droplets and monomer-swollen micelles. The particle growth stage ends when monomer droplets disappear in the polymerization system. In the fina l stage, the concentration of monomer in the reaction loci continues to decrease toward the end of the polymerization. 9

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Figure 2.2: Schematic representation of the micelle nucleation model39 I 2* M M M M M M M M MM MM M M M M M MPMPM MPPM MPMPM PPP Nucleation of monomer-swollen micelles Growth of latex particles Latex particles Monomer droplets Consumption of residual monomer M*I Initiator molecule Initiator radical Surfactant molecule Oligomeric radical Monomer molecule Polymer chain P 10

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Acrylates, or esters of acrylic acid, are suitable to be used in emulsion polymerization. Emulsion polymerization of several monomers including butyl acrylate, methyl methacrylate and methyl acrylate has been reported for the formation of nanoparticles.40 These monomers serve as the basis of these studies. 2.3 Synthesis of carbohyd rate acrylate monomers Carbohydrate monomers required for free-radical emulsion polymerization were prepared in the form of acrylates or acrylamides. Acrylation can be achieved at a sugar hydroxyl or amino group after protecting the other OH groups in the molecule. For the preparation of polymeric glyconanoparticles, a variety of structurally diverse monosaccharide acrylates derived from glucose, ribose, mannose, galactos e and glucosamine were synthesized as co-monomers (Figure 2.1). The following section describes the synthetic procedures for the preparation of these monomers from their rele vant commercially available monosaccharides. Figure 2.3: Carbohydrate monomers used for the formation of glyconanoparticles: 3-acryloyl-1,2:5,6-diO isopropylidene-D-glucofuranose ( 1 ), 1-acryloyl2,3:5,6-diO -isopropylidene-D-mannofuranose ( 2 ), 6acryloyl-1,2:3,4-diO -isopropylidene-D-galactopyranose ( 3 ), N -acryloyl 1,3,4,6-tetraO -acetyl-Dglucosamine ( 4 ), and 5O -acryloyl -2,3-isopropylidene-1-methoxy-D-ribofuranose ( 5 ). O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O N H O O O O O O O 1 3 4 5 2 11

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2.3.1 O -Isopropylidene protected carbohydrate acrylates 1-4 Carbohydrate acrylates derived from glucose, galactose, mannose and ribose were prepared by a twostep procedure (as shown in Scheme 2.1) vi a the corresponding acetonides. The diacetonides 6 and 7 Scheme 2.1: Synthesis of O -isopropylidene protected carbohydrate acrylates a b 6 1O OH OH HO HO HO O O O HO O O O O O O O O O D-glucose O OH OH HO HO HO D-mannose cO OH O O OO O O O O OO O O OH OH HO HOD-galactose OH O O O O O OH O O O O O O O O HOOH HO OH 4O O OO O O O O OO HO a b b d b 2 3 7 8 9 D-ribose Conditions: (a) acetone, CuSO4, H2SO4, (yields: 6 7 =82, 83% ); (b) acryloyl chloride, Et3N, CH2Cl2 (yields: 1 2,3,4 =68, 65, 62, 65% ); (c) acetone, ZnCl2, H3PO4, P2O5, 85% ;(d) acetone, 2,2-di methoxy propane, MeOH, HCl, 86%. 12

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were synthesized from glucose and galactose respectively using acetone with concentrated H2SO4 as catalyst.41 As the use of this procedure resulted in a low yield of compound 8 the acetonation of mannose was achieved with ZnCl2 in combination with H3PO4.42 2,2-Dimethoxypropane with acetone was used to prepare O isopropylidene derivative 9 from ribose.41 Acrylation of these acetonides with acryloyl chlorides and Et3N in CH2Cl2 afforded monomers 1 2 3 and 4 in high yields. 2.3.2 O -acetal protected acrylamide from glucosamine Scheme 2.2: Synthesis of N -acryloyl 1,3,4,6-tetraO -acetyl-D-glucosamine aO OH NH3Cl HO HO HO glucosamine hydrochloride O OH N HO HO HO OCH3 O OAc N AcO AcO AcO OCH3 b O OAc NH3Cl AcO AcO AcO O OAc HN AcO AcO AcO O c d 5 1011 12 Conditions: (a) aq NaOH, p -anisaldehyde,70%; (b) Py, Ac2O, 80%; (c) acetone, 5M HCl, 85%; (d) acryloyl chloride, Et3N, CH2Cl2, 65%. Compound 12 was obtained from commercially available D-glucosamine hydrochloride in a procedure 43 that used p -anisaldehyde for protection of the amine (compound 10 ), acetylation (compound 11 ) and removal of the p -methoxybenzylidene group with HCl in acetone (compound 12 ). Acrylation of this product with acryloyl chloride and Et3N in CH2Cl2 afforded compound 5 13

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2.4 Glyconanoparticle preparatio n by microemulsion polymerization Glyconanoparticles were prepared with a 9:1 (by weight) blend of ethyl acrylate and one of the monosaccharide acrylates (20% by wei ght) by emulsion polymerization (Sch eme 2.3). Sodium dodecyl sulfate (also known as sodium lauryl sulfate) was used as the surfactant for this preparation and potassium persulfate was used as the initiator as the polymerization is in an aqueous system. The structur ally diverse monosaccharide acrylates derived from glucose, galactose, mannose, ribose and glucosamine ( monomers 1 2 3 4 and 5 respectively) were used as co-monomers to prepar e the corresponding polymeric glyconanoparticles ( GNP1 GNP2 GNP3 GNP4 and GNP5 ). Briefly, ultrapure water and sodium dodecyl sulfate (SDS) were added to the mixture of monomers, and the mixture was pre-emulsified with stirring under a nitrogen atmosphere. A milky colored emulsion (Fig. 2.1) was prepared by adding potassium persulfate to the mixture and stirring at 70 oC for 6 h. The polymerization conditions are given in Table 2.1. Scheme 2.3: Preparation of glyconanoparticles by emulsion polymerization O O O O O +O O O O O O O O carbohydrate monomer ethyl acrylate emulsion polymerization glyconanoparticle 1, 2, 3, 4, 5 GNP1, GNP2, GN P3, GNP4, GNP5 Figure 2.4: A vial containing a representative carbo hydrate-bearing polyacrylate emulsion. 14

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Table 2.1 : Formulation used for the emulsion polymerization ___________________________________________________ Components Amounts Solid Content Monosaccharide acrylate 100 mg 9.6% Ethyl acrylate 900 mg 86.5% Sodium dodecyl sulfate 30 mg 2.9% Potassium persulfate 10 mg 1.0% Water 4 mL Reaction temperature 70 o C 2.5 Glyconanoparticle characterization The physicochemical properties of the glyconanoparticles were examined using scanning electron microscopy (SEM), dynamic light scattering (DLS) and 1H NMR spectroscopy. Emulsion polymerization of differentially O -protected monosaccharide monomers 1 5 showed no significant dependence on the nature of the monosaccharide or on the location of the acryloyl moiety. The following chapter describes the characterization of glycona noparticles prepared with 3O -acryloyl-1,2:5,6-di-O-isopropylidine-Dglucofuranose ( 1 ) using these techniques. 15

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2.5.1 Scanning Electron Microscopy (SEM) analysis The morphology and the particle size of the emul sified nanoparticles were examined by scanning electron microscopy (SEM). SEM measurements of th e glyconanoparticles were captured on a Hitachi S800 SEM instrument in the USF Department of Engineering. Samples were prepared by placing a drop of diluted emulsion (1 L of emulsion in 30 mL of de-ionized distilled water) on the silicon wafer and evaporating the solvent by air blowing prior to coating with gold sputter under high vacuum. The SEM image of glyconanoparticles prepared with 3O -acryloyl-1,2:5,6-diO -isopropylidene-D-glucofuranose ( 1 ) and ethyl acrylate ( GNP1 ) is shown in Figure 2.5. As seen in the imag e, the nanoparticles were essentially perfectly spherical and measured from 20 to 70 nm in diameter. Figure 2.5: SEM image of glyconanoparticles prepared from 3O -acryloyl-1,2:5,6-diO -isopropylidene-Dglucofuranose ( 1 ) and ethyl acrylate by emulsion polymerization ( courtesy: Dr. J. Y. Shim). 16

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2.5.2 Dynamic Light Scattering (DLS) analysis DLS analyses were performed using a UPA 150 Honeywell MicroTrac DLS instrument equipped with a laser beam at 780 nm. The sample was diluted with de -ionized distilled water (0.05 mL of emulsion in 9.95 mL of water, polymer concentration 0.1% wt) and sonicated for 3 minutes for the analysis. The value is expressed in weight-averaged scales as unimode at a scattering angle of 180 o (backscatter) at a temperature of 25 oC. The particle size distribution of GNP1, obtained by DLS analysis, is shown in Figure 2.6. The average diameter of the glyconanoparticles was 42 nm, with a statistical standard deviation of 9 nm, and the particles measured from 15 to 70 nm in diameter. Table 2.2 summ arizes the particle size for each one of the polyacrylate glyconanoparticles GNP 1-5 Figure 2.6: Particle size distribution of glyconanoparticles prepared from 3O -acryloyl-1,2:5,6-diO isopropylidene-D-glucofuranose ( 1 ) and ethyl acrylate by emulsion polymerization. Table 2.2 : Average particle sizes of glyconanoparticles GNP 1-5 Glyconanoparticles Average Particle Size (nm) GNP1 GNP2 GNP3 GNP4 GNP5 42.3 40.6 45.1 35.2 44.1 17 wt % Diameter (nm)

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2.5.3 1H NMR analysis 1H NMR spectroscopic analysis of each nanoparticle material was performed using a Bruker DPX-250 spectrometer. To prepare the sample, a film was formed by evaporation of 0.15 ml of the polymer emulsion spread on a clean glass surface. The f ilm was dried at room temperature for 24 hrs, and then taken up (swelled) in CDCl3 to perform the NMR experiment (Figure 2.7) Figure 2.7: Typical 1H NMR spectra of (a) 3O -acryloyl-1,2:5,6-diO -isopropylidine-D-glucofuranose ( 1 ) and (b) the glycopolymer obtained from the emulsion after the film formation. 18 O O O O O O O (a) (b) H-1 H-3 H-2 O O O O O O O O O

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Figure 2.7a shows the original 1H NMR spectrum of the carboh ydrate acrylate co-monomer, 3O -acryloyl1,2:5,6-diO -isopropylidene-D-glucofuranose ( 1 ), while Figure 2.7b shows the 1H NMR spectrum of the glycopolymer obtained after polymerization. Notably, the signals of the acryloyl protons (6.42-5.92 ppm in Figure 2.7a) have completely disappeared after the polymerization and broad absorptions for the protons H-1, H-3 and H-2 (5.90, 5.17 and 4.36 ppm in Figure 2.7b) of the monosaccharide moiety are plainly visible. This confirms the complete conversion of the carbohydrate monomer to polyacrylate within the nanoparticle matrix, without any detectible amount of residual unreacted monomer. 2.6 Influence of bulky aryl protecting groups in the carbohydrate acrylate monomer on glyconanoparticle preparation. Upon the successful execution of the radical emulsion polymerization process using protected manosaccharides 1-5 these studies were extended to two other O -protected carbohydrate monomers, O -benzyl and O -benzoyl protected monosaccharide acrylates 13 and 14 (Figure 2.8). These tw o carbohydrate acrylate monomers bearing bulky aryl protecting groups were synthesized as shown in Schemes 2.4 and 2.5. Figure 2.8: 1O -Acryloyl-2,3,5-triO -benzyl-D-ribofuranose ( 13 ) and 2N -acryloyl-1,3,4,6-tetraO -benzoyl-D-glucosamine ( 14 ). O OBz N H O OBz BzO OBz O OBn BnO BnO O O 13 14 2.6.1 1O -Acryloyl-2,3,5-triO -benzyl-D-ribofuranose (13) Scheme 2.4: Synthesis of 2,3,5-triO -benzyl-D-ribofuranose-1-acrylate a D-ribose O HOOH HO OH O O BnOOBn BnO O OH BnOOBn BnO O O BnOOBn BnO O b c 13 15 16 Conditions: (a) i. H2SO4, methanol; ii. BnBr, NaH, DMF, 77%; (b) 0.1N HCl, Dioxane, 65%; (c) acryloyl chloride, Et3N, CH2Cl2, 60%. 19

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The synthesis of compound 13 started with D-ribose as shown in sche me 2.4. Using methanolic sulfuric acid, the free sugar (D-ribose) was converted to methyl -D-ribofuranoside44 which afforded methyl 2,3,5-triO benzyl-D-ribofuranoside ( 15 ) after benzylation with BnBr in DMF-NaH.45 The product was hydrolyzed in dioxane solution with aqueous hydrochloric acid to give 2,3,5-triO -benzyl-D-ribose44 ( 16 ). Acrylation with acryloyl chloride and Et3N in CH2Cl2 then afforded compound 13 2.6.2 N -Acryloyl-1,3,4,6-tetraO -benzoyl-D-glucosamine (14) Scheme 2.5: Synthesis of N -acryloyl-1,3,4,6-tetraO -benzoyl-D-glucosamine O OBz N BzO BzO BzO OCH3 a O OBz NH3Cl BzO BzO BzO O OBz HN BzO BzO BzO O b c 10 14 17 18 Conditions: (a) BzCl, Py, 71%; (b) acetone, 5M HCl, 87%; (c) acryloyl chloride, Et3N, CH2Cl2, 63%. As shown in scheme 2.5, compound 14 was synthesized from the intermediate product 10 after benzoylation (compound 17 ), removal of the p -methoxybenzylidene group with HCl in acetone (compound 18 ) and acrylation with acryloyl chloride and Et3N in CH2Cl2. To my surprise, 1H NMR analysis revealed that the use of th ese monomers was not as successful in the preparation of glyconanoparticles as those from acrylates 1-5 as significant amounts of the unreacted carbohydrate acrylates remained after polymerization. The reasons for this outcome are not obvious, but the presence of these bulky aryl protecting groups on the suga r monomer may affect its reactivity or radical transfer capabilities, and thus induce low incorporation into the polymer backbone. 20

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2.7 Influence of free hydroxyl gr oups of the monosaccharide acryla te monomer on the formation of glyconanoparticles. In direct follow-up to this last set of experiments, additional studies were carried out to investigate whether a carbohydrate acrylate having free (unprotected) hydroxyl groups could be used directly for the nanoparticle formation. This was first studied with 3O -acryloyl-D-glucose ( 19 ) (Scheme 2.6), the deprotected form of acrylate 1 prepared by treating compound 1 with 90% trifluoroacetic acid. The emulsion prepared with 19 and ethyl acrylate as a co-monomer appeared much less transparent to the eye than the emulsion prepared with the relevant protected monomer, 3O -acryloyl-1,2:5,6-diO -isopropylidene-D-glucofuran-ose ( 1 ), using the same formulation. In fact, DLS analysis of the emul sion indicated the average particle size was around 85 nm in diameter, about double the size of the particles prepared from the O -protected acrylate 1 We obtained similar results with the emulsions prepared with 5O -acryloyl-1-methoxy-D-ribofuranose ( 20 ) (Scheme 2.6) versus 5O -acryloyl-2,3O -isopropylidene-1-methoxy--D-ribofuranose ( 5 ) Scheme 2.6: Deacetonation of carbo hydrate acrylates 1 and 5 O OH HO O O O 20O OH OH O HO HO O 19O O O O O O O 1O O O O O O 5 a b Conditions: (a) 90% CF3COOH, rt, 30 min., 90%; (b) 80% CH3COOH, 80 oC, 2 hrs, 68% The photographic images in Fig. 2.9a show the increasing turbidity of the emulsions as the concentration of acrylate monomer 20 is increased from 5% (E2) to 7.5% (E3) to 10% (E4) of the monomer content, relative to the appearance of the emulsion E1 prepared with fully protected monomer 5 (having 10% carbohydrate by weight relative to ethyl acrylate). Dynami c light scattering data confirms that this increasing cloudiness in the emulsions is due to larger glyconanoparticle formation (Fig. 2.9b) as the amount of unprotected carbohydrate monomer is increased from 5% (E2) to 10% (E4). This suggests that nanoparticle size may be dependent on the presence and the degree of hydrogen-bonding effects within the carbohydrate moiety while the polymeric matrix is being formed. 21

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Figure 2.9: Comparison of the effects of using an O -protected versus O -unprotected monosaccharide acrylate on nanoparticle size. (a) Photographs of four different emulsion samples prepared with 20% (by weight) polymer content from 5O -acryloyl-2,3O -isopropylidene-1-methoxy--D-ribofuranose ( 5 ) and 5O -acryloyl-1methoxy-D-ribofuranose ( 20) as co-monomers with ethyl acrylate. E1: monomer 5 and ethyl acrylate (1:9 by weight). E2: monomer 20 and ethyl acrylate (1:19 by weight). E3: monomer 20 and ethyl acrylate (1:14 by weight). E4: monomer 20 and ethyl acrylate (1:9 by weight); (b) Av erage particle sizes of these formulations from DLS analysis. 22 E1 E2 E3 E4 (b) 0 10 20 30 40 50 60 70 80 90 E1E2E3E4Average particle size (nm)(a)

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2.8 Nucleoside-bearing po lyacrylate nanoparticles Nucleosides are a well-studied class of drugs useful for treating viral infections and leukemia. In most cases, inhibition of viral replication and cell prolifera tion is associated with intracellular conversion of nucleosides to nucleoside triphosphates, which act as either inhibitors of viral and cellular DNA and RNA polymerases or as chain terminators following incorporation into a growing DNA or RNA strand46. Experiments were done to investigate whether nucleoside-bearing polyacrylate nanoparticles could be prepared for potential further biological evaluati on, using acrylated cytidine analogue 23 (Scheme 2.7). 2.8.1 4N -Acetyl-5’O -acryloyl-2’,3O -isopropylidenecytidine Scheme 2.7: Synthesis of 4N -acetyl-5’O -acryloyl-2’,3O -isopropylidenecytidine a b CytidineO OO HO O N HOOH HO N O NH2 N N O NH2 O OO HO N N O H N O O OO O N N O H N O O c 21 22 23 Conditions: (a) i. p -TsOH, acetone ii. aq. NaHCO3, 60%; (b) Ac2O, methanol, reflux, 86%; (c) acryloyl chloride, Et3N, CH2Cl2, 60%. 2’,3’O -Isopropyliden e cytidine ( 21) was prepared from cytidine using p -toluenesulfonic acid in acetone.47 Treatment of compound 21 with acetic anhydride in methanol afforded 4N -acetyl-2’,3’O -isopropylidene cytidine48 ( 22 ) which was acrylated to give compound 23 Monomer 23 was polymerized in microemulsion and cytidine functionalized polyacrylate nanoparticles were prepared. 23

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2.9 Conclusions In this chapter, the first preparation of polyacryl ate nanoparticles bearing carbohydrates within the polymeric framework by emulsion polymerization was demonstrated. The glyconanoparticles displayed narrow size distributions in the range of 20-70 nm. Acetonide and acetyl protected monosaccharide acrylates were found to be well suited for this preparation as proton NMR analysis of the relevant emulsions indicated essentially 100% incorporation of the carbohydrate acrylate monomer into the polymer. An increase of the particle size was observed upon use of 3O -acryloyl-D-glucose ( 19 ) and 5O -acryloyl-1-methoxy-Dribofuranose ( 20 ) as monomers bearing free hydroxyl groups. This methodology allows for a new, simple route to the synthesis of polymeric glyconanoparticles with potential applications in drug delivery and materials development. Also nucleosides can be used for this preparation and it may open potential avenues to using nucleoside-bearing nanoparticles as nucleic acid transporters.49 The next chapter will describe the use of these glyconanoparticles as drug delivery vehicles for various antibiotics and their biological activity. 24

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CHAPTER III GLYCONANOBIOTICS: SYNTHESIS AND BIOLOGICAL ACTIVITY 3.1 Introduction The use of nanoparticles in medicine is one of the important directions that nanotechnology is taking at this time. Their application in drug delivery,50-52 cancer cell diagnostics,53-56 and therapeutics57 have been active fields of research. The use of nanoparticles as drug delivery systems is of particular interest because they have some advantages such as easy purification and steriliza tion, drug targeting possibilities, and sustained release action.58-59 However, the use of nanoparticles as drug delivery vehicles in the field of antibiotic therapy has received relatively little attention. 60-61 The Turos group recently reported the formation of antibiotic-conjugated polyacrylate nanopaticles as a water solubility enhancer for antibiotics having extremely low water solubility. In this process, the antibiotic is converted to an acrylated derivative with the reaction of acryloyl chloride and then used as a co-monomer with styrene/butyl acrylate mixture in microemulsion polymerization. To dissolve the antibiotic drug monomer in the styrene/butyl acrylate co-monomeric mixture to homogeneity is an important step of this preparation to avoid incomplete drug incorporation into the polymer backbone. N -Thiolated lactam acrylated monomers such as 24 (Fig. 3.1) were found to be well suited to prepare nanoparticle antibiotics, or nanobiotics, by emulsion polymerization. However, when Dr. Suresh Reddy and Kerriann Greenhalgh in the Turos group tried to extend this protoc ol to other classes of antibiotics, such as penicillin and ciprofloxacin, the corresponding monomers ( 25 and 26 in Fig. 3.1) obtained from the reaction of acryloyl chlorides showed extremely lo w solubility in styrene/butyl acrylate. This resulted in much of the drug monomer being unreacted after the polymerizati on. Hence, it was felt that more li pophilic drug monomers are needed for this preparation. Figure 3.1: Structures of acrylated antibiotic monomers of N -thiolated -lactam ( 24 ), penicillin ( 25 ) and ciprofloxacin ( 26 ). N S H N O COOH O N N N O HOOC F O O N O Cl SCH3 O 24 25 26 25

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Although monosaccharides are well known for thei r hydrophilicity due to the presence of free hydroxyl groups, their protected forms are hydrophobic. The conjugation of an acrylated hydrophobic moiety to the lipophilic antibiotic will make it even more lipophilic and suitable as a co-monomer in microemulsion polymerization. This idea led to the aim of preparing novel carbohydrate-based acrylated acyl chlorides to synthesize carbohydrate conjugated drug monomers. This chapter describes the synthesis of novel carbohydrate-based reagents to prepare carbohydrate-conjugated drug monomers, and the microemulsion polymerization of these monomers to pr epare glyconanoparticle antibiotics, or glyconanobiotics The biological activity of the drug monomers and the glyconanobiotics derived from them is also discussed. 3.2 Synthesis of novel carbohydrate-based acyl chlorides For these exploratory investigations, tw o glucose-based acrylated acyl chlorides ( 27 and 28 ) shown in Fig. 3.2 were synthesized and used in place of acryloyl chloride to pr epare carbohydrate-conj ugated antibiotic monomers for the emulsion polymerization (to be described). Figure 3.2: Structures of diacyl chloride 27 and acyl chloride 28 28O O O O O O O O O Cl O 27O O O O O O O O Cl O O Cl O 26

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3.2.1 3O -Acryloyl-1,2O -isopropylidene-5,6 bis((chlorosuccinyl)oxy)-D-glucofuranose (27) The first of these acrylated acid chlorides, compound 27 was prepared in three steps from protected glucose acrylate 1 (Scheme 3.1). Scheme 3.1: Synthesis of 3O -Acryloyl-1,2O -isopropylidene-5,6 bis((chlorosuccinyl)oxy) -D-glucofuranose ( 27 ) 1O O O O O O O 29O O O O HO HO O 30O O O O O O O O OH O O HO O 27O O O O O O O O Cl O O Cl O a b c Conditions: (a) 80% CH3COOH, 80 oC, 4h, 62% ; (b) succinic anhydride, Et3N, CH2Cl2, 25 oC, 5h, 65%; (c) SOCl2, 25 oC, 3h, 98%. Partial deacetonation of acrylate monomer 1 with 80% acetic acid at 80 oC and conjugation of the product 29 with succinic anhydride in the presence of Et3N and dry dichloromethan e afforded the diacid 30 in 40% overall for the two steps. Thionyl chloride was used to convert compound 30 to the corresponding diacyl chloride 27 While diacid 30 was characterized by 1H NMR, diacyl chloride 27 was used without purification. 27

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3.2.2 6O -Acetyl-3O -Acryloyl-1,2O -isopropylidene-5-((chlorosucciny l)oxy)-D-glucofuranose (28) Scheme 3.2 describes the synthesis of acrylated acyl chloride 28 Selective acylation of the primary hydroxyl group of the diol62 29 was achieved in 72% yield with a cetyl chloride in dichloromethane in the presence of 2,4,6-collidine to give compound 31 Acyl chloride 28 was obtained in two steps via acid 32 in a similar procedure to that described in Scheme 3.1. Scheme 3.2: Synthesis of 6O -acetyl-3O -acryloyl-1,2O -isopropylidene-5-((chlorosuccinyl)oxy)-Dglucofuranose ( 28 ) 31O O O O HO O O O 28O O O O O O O O O Cl O a 32O O O O O O O O O HO O b c 29 Conditions: (a) collidine, AcCl, CH2Cl2, -40 oC, 3h, 72%; (b) succinic anhydride, Et3N, CH2Cl2, 25 oC, 5h, 64%; (c) SOCl2, 25 oC, 3h, 98%. 3.3 Synthesis of novel antibiotic-conjugated carbohydrate monomers 3.3.1 N -Thiolated -lactam-conjugated glucose acrylate (34) N -Thiolated -lactams discovered in the Turos group ar e a new family of antibacterial agents active against Staphylococcus bacteria, including methicillin-resistant strains of Staphylococcus aureus (MRSA) 63-64, and Bacillus anthracis 65 which is the causative agent of anthrax infections. These compounds have a mode of action distinct from that of all other -lactam antibiotics.63 Rather than interfering directly with cell wall biosynthesis th rough irreversible acylation of penicillin binding transpeptidases, these co mpounds seem to affect cellular pr ocesses through transfer of the N -organothio group to a bacterial thiol.65 For the goal of preparing nanoparticle variants of this class of antibacterial agents, bisN -thiolated lactam glucose acrylate 34 was selected as a target. 28

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Monomer 34 was synthesized from N-sec -butylthio -lactam derivative66 33 by coupling with diacyl chloride 27 and Et3N in CH2Cl2 as shown in Scheme 3.3. The yield for the coupling was 62%. 1H NMR and ESI-MS spectra of compound 34 are shown in Fig. 3.3 and Fig. 3.4 respectively. Scheme 3.3: Synthesis of N-sec -butylthio -lactam monomer 34 O O O O O O O O O O O O O N N O Cl S O S Cl O O O O O O O O Cl O O Cl O Et3N, CH2Cl2HO N O Cl S 33 34 62% 27 29

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Figure 3.3: 1H NMR spectrum of N-sec -butylthio -lactam monomer 34 Figure 3.4: ESI-MS spectrum of N-sec -butylthio -lactam monomer 34 (m/z 1028.3 [M+NH4]+, Calcd. 1028.0) 30

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3.3.2 Ciprofloxacin-conjugated glucose acrylates (36 and 37) Anthrax, an infectious disease caused by the bacterium Bacillus anthracis poses a significant threat to the public as a bioterrori st agent. Ciprofloxacin is a fluoroquinolone antibiotic that is commonly used to treat anthrax infections. Its mode of action depends upon blocking bacterial DNA replication by binding to an enzyme called DNA gyrase, thereby causing double-stranded breaks in the bacterial chromosome.67 In concert with the previously stated objec tive of preparing antibiotic-conjugated polyacrylate nanoparticles, bis-ciprofloxacin and mono-ciprofloxacin glucose acrylates 26 and 27 respectively, were chosen as potential monomers for the emulsion polymerization. Ciprofloxacin-conjugated monomers 36 and 37 were synthesized by coupling with diacyl chloride 27 and acyl chloride 28 respectively and Et3N in CH2Cl2 (Schemes 3.4 and 3.5). The coupling o ccurred in over 50% yield in each case. Scheme 3.4: Synthesis of bis-ciprofloxacin monomer 36 O O O O O O O O O O O N N N O HOOC F N N N O COOH F O O O O O O O O O O O NH2Cl N N O HOOC F Cl Cl Et3N, CH2Cl235 36 50% 27 31

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Scheme 3.5: Synthesis of mono-ciprofloxacin monomer 37 O O O O O O O O O O N N N O HOOC F O O O O O O O O O O Cl NH2Cl N N O HOOC F Et3N, CH2Cl235 37 56% 28 1H NMR and ESI-MS spectra of compound 37 are shown in Fig. 3.5 and Fig. 3.6 respectively. 32

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Figure 3.5: 1H NMR spectrum of mono-ciprofloxacin monomer 37 Figure 3.6: ESI-MS spectrum of mono-ciprofloxacin monomer 37 (m/z 752.2 [M+Na]+, Calcd. 752.6) 33

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3.3.3 Penicillin-conjugated glucose acrylate Penicillin is one of the earliest discovered and widely used antibiotic agents, derived from the Penicillium mold. The antibacterial effect of penicillin was discovered by Sir Alexander Fleming in 1929. Penicillin derivatives produce their bacteriocidal effects by inhibiting the formation of peptidoglycan cross links in the bacterial cell wall. 68 The -lactam moiety of penicillin binds to the enzyme (transpeptidase) that links the peptidoglycan molecules in bacteria, and this weakens the cell wall of the bacterium when it multiplies (in other words, the antibiotic causes cytolysis or death when the bacterium tries to divide). Although -lactam antibiotics such as penicillin G have been mainstays of clinical treatment for many types of bacterial infections, their effectiveness has been compromised by the ability of drug-resistant bacteria to produce -lactamase enzymes th at hydrolyze the -lactam moiety in these antibiotics to render them inactive. The studies in the Turos lab have revealed that penicillin-bound polyacrylate nanoparticles prepared by microemulsion polymerization are able to recover the in vitro antibacterial activity of penicillin against -lactamase-producing MRSA.69 Penicillin-conjugated monomer 40 was synthesized from 6-aminopenicillanic acid ( 38 ) via its trimethylsilyl ester 39 which was then reacted w ith acyl chloride to give 40 in 51% yield (Scheme 3.6). 34

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Scheme 3.6: Synthesis of penicillin-c ontaining acrylate monomer 40 O O O O O O O O O O O O O O O O O O O O Cl Et3N, CH2Cl2N S H2N O COOTMS N S H N O HOOC N S H2N O COOH N,O-Bis(trimethylsilyl) acetamide, CH2Cl239 40 38 51% 28 35

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3.4 Glyconanobiotics by emulsion polymerization Scheme 3.7: Preparation of glyconanobiotics by emulsion polymerization O O O O O O O drug monomer butyl acrylate emulsion polymerization glyconanobiotic 34, 37, 40 GNB34, GNB37, GNB40 antibiotic drug styrene + + O O O antibiotic drug O O O antibiotic drug Glyconanoparticle antibiotics, or glyconanobiotics, were prepared from the above-described antibiotic-conjugated carbohydrate monomers and a blend of styrene/butyl acrylate (3:7 w/w) by microemulsion polymerization using sodium dodecyl sulfate (3% w/w) and potassium persulfate (1% w/w) as the surfactant and the initiator, respectively (Schem e 3.3). The general procedure involved dissolving the drug monomer in the mixture of styrene/butyl acrylate and pre-emulsifying the mixture in purified water containing sodium dodecyl sulfate with stirring under a nitrogen atmosphere. Then the emulsion was prepared by adding potassium persulfate to the mixture and stirring at 70 oC for 6 hours. The polymerization conditions used to prepare GNB34 GNB37 and GNB40 are given in Table 3.1. GNB 36 could not be made, however, because the bis-ciprofloxacin monomer 36 was not soluble in the mixture of styrene/butyl acrylate. 36

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Table 3.1 : Formulation used for the emulsion polymerization to prepare antibiotic-conjugated glyconanoparticles GNB 34 GNB 37 and GNB 40 ___________________________________________________ Components Amounts Solid Content Drug monomer 20 mg 2% Butyl acrylate 658 mg 65.8% Styrene 282 mg 28.2% Sodium dodecyl sulfate 30 mg 3% Potassium persulfate 10 mg 1% Water 4 mL Reaction temperature 70 o C These glyconanobiotics displayed a highly controlled particle diameter of about 40 nm, and possessed antibacterial activity by virtue of having an antibiotic compound covalently bound within the matrix. It is curious that under these conditions the particle size is not significantly changed even though the properties of the antibiotic monomers are dramatically different. Table 3.2 summarizes the particle size for each prepared glyconanobiotic GNP 34, GNP37 and GNP40 Table 3.2 : Average particle sizes of glyconanoparticles GNP 34, GNP37 and GNP40 as determined by dynamic light scattering. Glyconanobiotic Average Particle Size (nm) GNB34 GNB37 GNB40 38.9 45.1 43.4 3.5 Biological activity The antimicrobial activity of the antibiotic-conjugated carbohydrate monomers and their corresponding emulsions (glyconanobiotics) were determined by measuring the minimum inhibitory concentration, or MIC, values in broth media. MI C determination is an accepted method for assessing antimicrobial activity of compounds. This method is an aqueous phase technique where bacteria, nutrients and antibiotic candidates are all suspended in solution together. The minimum amount of compound required to completely inhibit visible bacterial grow th is the MIC value for that compound. The lower the MIC, the stronger th e compound’s bioactivity. The bacterial growth is examined optically for changes in opacity due to increasing cell counts. The monomers and the emulsions were indivi dually tested for antibacterial activity against Staphylococcus aureus ( S. aureus ), a strain of methicillin-resistant Staphylococcus aureus (MRSA) and Bacillus anthracis ( B. anthracis ). S. aureus and MRSA microbes were purchased from American Type Culture Collections (ATCC). 37

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3.5.1 Biological activity of antibiotic monomers 34 36, 37, and 40 against S. aureus MRSA and B. anthracis The minimum inhibitory concentration (MIC) values for the antibiotic monomers 34 36 37 and 40 are summarized in Table 3.2. Since compound 34 was not readily miscible in broth media, the MIC values were obtained by agar plate dilution. For the monomers 36 37 and 40 the MIC values were obtained in broth media. N-Thiolated -lactam-conjugated acrylate 34 and ciprofloxacin-conjugated acrylates 36 and 37 showed significant activity against S. aureus MRSA and B. anthracis while the penicillin monomer 40 had no in vitro activity against these three microbes. Bis-ciprofloxacin monomer 36 showed higher activity against S.aureus and MRSA than the mono-ciprofloxacin monomer 37 whereas the respective activities against B. anthracis were reversed. Table 3.3 : MIC values for antibiotic-conjugated carbohydrate monomers Monomer S.aureus (ATCC 25923) MRSA (ATCC 43300) B. anthracis 34 36 37 40 126 nM 30 nM 44 nM > 393 nM 126 nM 30 nM 44 nM > 393 nM 63 nM 60 nM 22 nM > 393 nM 3.5.2 Biological activity of glyconanobiotics GNB 34, GNB 36, and GNB 40 against S. aureus MRSA and B. anthracis The same in vitro experiments were run for GNB 34 GNB 36 and GNB 40 MIC values of these glyconanobiotics are shown in Table 3. 3 and the inactive penicillin monomer 40 proved to generate inactive glyconanobiotics after polymerization Table 3.4 : MIC values for glyconanobiotics The glyconanobiotic GNB 36 showed the same activity profile against S. aureus MRSA and B. anthracis as the relevant ciprofloxacin monomer 36 The N-thiolated -lactam glyconanobiotic GNB 34 tested in broth media showed higher activity against S. aureus and MRSA than the -lactam monomer 34 38 Glyconanobiotic S.aureus (ATCC 25923) MRSA (ATCC 43300) B. anthracis GNB 34 GNB 36 GNB 40 63 nM 44 nM > 393 nM 63 nM 44 nM > 393 nM 63 nM 22 nM > 393 nM

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3.6 Conclusions Novel carbohydrate-based acrylated acyl chlorides 27 and 28 were synthesized from glucose and used in place of acryloyl chloride to pr epare carbohydrated antibiotic drug monomers 34 36 37 and 40 These antibiotic-conjugated carbohydrate monomers were polymerized in microemulsion with styrene/butyl acrylate to prepare glyconanoparticle antibiotics (glyconanobiotics). The monomers and corresponding glyconanobiotics showed biological activity against S. aureus MRSA and B. anthracis microbes. This development of antibiotic-laden glyconanoparticles demonstrates for the first time the use of the polymeric glyconanoparticles as drug delivery vehicles. The next chapter will discuss the use of these glycosylated nanoparticles as recognition units in carbohydrate ligand me diated targeted drug delivery. 39

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CHAPTER IV PREPARATION OF POLYACRYLATE GLYCONANOPARTICLES WITH SURFACE CARBOHYDRATES 4.1 Introduction Glycotargeting, the use of car bohydrate ligands to bind selec tively to protein receptors, is a promising approach in targeted drug delivery. Glycotargeting relies on carrier molecules possessing carbohydrates that are recognized and internalized by cell surface mamma lian lectins. Numerous types of glycotargeting vehicles have been de signed based on the covalent attach ment of saccharides to proteins, polymers and other aglycones. These carriers have fo und their major applications in antiviral therapy, immunoactivation, enzyme replacem ent therapy and gene therapy.70 To investigate the use of polyacrylate glycona noparticles in ligand-mediated targeted drug delivery as shown in Fig. 4.1, study was undertak en to prepare and characterize new polyacrylate nanoparticles whose surfaces are modified with carbohydrates. The binding ability of these glyconanoparticles was evaluated by fluorescence spectroscopy with the use of sugar-binding boronic acids. This chapter describes the preparation of two glucose-functionalized polyacrylate nanoparticles as prototypes (Fig. 4.2), using emulsion polymerization as described in the previous chapter, and the characterization of the physical properties. Figure 4.1: A cartoon showing the use of ca rbohydrate ligands to target prot ein receptors (curly structures) in targeted drug delivery to bacterial cell. 40 O O Drug Drug

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Figure 4.2: Glucose-functionalized polyacrylate nanoparticle s with (a) short and (b) long tether lengths. O OH HO O HO HO O O O O O O O OH HO O HO HO (a) (b) GNP 19 GNP 41 4.2 Preparation of acrylated glucose monomers Two acrylated glucose monomers with different tether lengths (Fig. 4.3) were used to prepare these glyconanoparticles by microemulsion polymerization with styrene/butyl acrylate. Monomer 19 3-acryloyl-Dglucose, can be prepared by deacetonation of monomer 1 as previously described in scheme 2.6. Monomer 41 with a long-chain acrylic moiety, was synthesized from acrylic acid as shown in scheme 4.1. Figure 4.3: Acrylated D-glucose monomers O OH OH O HO HO O O O O O O O OH OH O HO HO 19 41 41

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Scheme 4.1: Synthesis of monomer 41 O O O O O O O O O O O OH O ONa O O O O O O OH OH O O O O OH OH O HO HO OO O O a b c d e 41 46 45 44 43 42 Conditions: (a) NaOH 90 %; (b) 11-bromoundecan-1-ol, n-tetrabutylammonium bromide, 2,6-di-t-butyl-4methylphenol, water, chloroform, 100 oC, 3 days, 58%; (c) succinic anhydride, Et3N, CH2Cl2, 25 oC, 5h, 80%;(d) diacetone-D-glucose, EDCI, DMAP, CH2Cl2, 75%; (e) 90% CF3COOH, 30 min., rt, 67%. 11-Acryloyloxy undecan-1-ol ( 44 ) was prepared from acrylic acid ( 42 ) according to a procedure 71 that used 1 eq. of sodium hydroxide to prepare sodium acrylate ( 43 ) which was reacted with 11 -bromoundecan-1-ol to give compound 44 Conjugation of the product 44 with succinic anhydride in the presence of Et3N and dry dichloromethane afforded the acrylated acid 45 and compound 46 was obtained from EDCI coupling of the product 45 with diacetone-D-glucose. Deacet onation of compound 46 with 90% tr ifluoroacetic acid afforded the acrylate 41 4.3 Microemulsion polymerization of acrylated glucose monomers Glycosylated polyacrylate nanoparticles, bearing free sugar hydroxyl groups, were prepared with a 9:1 (by weight) blend of styrene/butyl acrylate and one of the monomers (20% by weight) by emulsion polymerization (Scheme 4.1). The polymerization conditions are given in Table 4.1 and the same emulsion polymerization procedure was used for this preparation as for the preparations of glyconanoparticles in chapter II and glyconanobiotics in chapter III using sodium dodecyl sulfate and potassium persulfate as the surfactant and initiator, respectively, in nano-pure water at 70 oC. 42

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Scheme 4.2: Preparation of glyconanoparticles with surface carbohydrates by emulsion polymerization. GNP 19, GNP 41 19, 41 emulsion polymerization O O Table 4.1 : Formulation used for the emulsion polymerizat ion to prepare glyconanoparticles with surface Carbohydrates ___________________________________________________ Components Amounts Solid Content Carbohydrate monomer 100 mg 9.6 % Butyl acrylate 630 mg 60.5 % Styrene 270 mg 25.9 % Sodium dodecyl sulfate 30 mg 3 % Potassium persulfate 10 mg 1 % Water 4 mL Reaction temperature 70 o C The monomers having a short-chain acrylic moiety ( 19 ) and a long-chain acrylic moiety ( 41 ) were polymerized in microemulsions to prepare glyconanoparticles GNP 19 and GNP 41 GNP 19 is less transparent than GNP41 and dynamic light scattering analysis revealed larger nanoparticles for GNP 19 as shown in Table 4.2. The formation of larger nanoparticles could be due to the swelling of polymer as a result of hydrogen-bonding. 43

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Table 4.2 : Average particle sizes of glyconanoparticles Glyconanoparticle Average Particle Size (nm) GNP 19 GNP 41 66.2 34.1 4.4 Detection of the binding ability of glyconanoparticles After the preparation of glyconanoparticles GNP 19 and GNP 11 from monomers 19 and 41 the surface availability of the carbo hydrate moiety to bind with an external molecule wa s studied for the potential use of these glyconanoparticles as recognition units in carbohydrate-ligand mediated targeted drug delivery. Sugar-binding boronic acids were used for this purpose, as described in the following sections. 4.4.1 Sugar detection by boronic acids The use of boronic acids is regarded as one of the most reliable approaches for the detection and quantification of free carbohydrates in water. 72-75 The equilibrium involved be tween the boronic acid and carbohydrates is shown in Scheme 4.2. Boronic aci ds are electron deficient Lewis acids having a sp2-hybridized boron atom with a planar trigonal conformation.76 Boronic acids form fast and reversible covalent interactions with soluble carbohydrates. This complexation involves the coordination of vicinal diol groups to the boronate, causing displacement of the two alkox y ligands on boron to make a new boronate derivative. In water, this species exists preferably as an anionic boronate. The an ionic boronate is highly electron rich and possesses an sp3-hybridized boron atom with a tetrahedral conformation (Scheme 4.2). When the boronic acids are conjugated with a fluorophore, the neutral form of the boron group acts as an electron-withdrawing group while the anionic form acts as an electron-donating group. 77 This change in the electro nic properties of the boron group leads to spectral changes of th e fluorophore, a phenomenon that has been used for the development of fluorescent carbohydrate sensors.77-78 Hence, water-soluble boronic acids that change their fluorescence properties upon sugar binding are especially useful as reporter units as sensors for carbohydrates. Also, carbohydrate-sensitive boronic acids can be used as quenchers to modul ate the emission of a variety of dyes have been developed. These quencher/dye combinations w ould thus serve as signal transducers for the detection and quantification of sugars. 44

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Scheme 4.3: Equilibrium established between the aryl boronic acid and a carbohydrate; and its dependency on pH.79 B OH OH R O HO HO OH OH OH + 2H2O B R O O O OH OH OH B R O O O OH OH OH OH pKa ~ 6.1 OH pKa ~ 8.8O HO HO OH OH OH + 2H2O B OH R HO OH HO The detection of carboh ydrate functionality on the surface of glyconanoparticles GNP 19 and GNP 41 was attempted for the purpose of showing that carbohydrate moieties built into the polyacrylate backbone occupy at least some of the surface area of the nanoparticle, and that it ma y be possible to u ltimately quantify this. These studies were done by utilizing the combina tion of 4,4’-N,N-bis(benzyl-2-boronic acid)-bipyridinium dibromide (o-BBV, 48 ) and (trisodium 8-hydroxy-1,3,6-pyrenetrisulfonate) pyranine ( 47 ) (Figure 4.4). o-BBV is a boronic acid modified quencher of pyranine 47 a commercially available water soluble, anionic dye that can be excited by visible light and fluoresces with high quantum yield. 80 This quencher/dye combination operates in aqueous solution at pH 7.4, and has been reported to be highly sensitive in detecting glucose in aqueous media in the range of 0-1800 mg/dL (glucose concentration). 81 Figure 4.4: Pyranine ( 47 ) and o-BBV ( 48 ) SO3 -O3S -O3S OH N N B B HO OH OH HO 3Na 2Br 47 48 45

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4.4.1.1 4’,4’-N’,N’-bis(benzyl-2-boronic acid)-bipyridinium dibromide (o-BBV) o-BBV ( 48 ) was synthesized in two steps from o-tolylboronic acid ( 49 ) as shown in Scheme 4.2. Radical-induced bromination of compound 49 was carried out using N-bromosuccinimide in the presence of 2,2’-azobisisobutyronitrile (AIBN) in carbon tetrachloride to give o-bromomethylphenylboronic acid82 ( 50 ) which afforded o-BBV ( 48 ) after refluxing with anhydrous methanol (to prepare dimethyl-(2-bromomethyl)benzeneboronate intermediate) an d reacting with 4,4’-dipyridyl.81 Scheme 4.4: Synthesis of 4’,4’-N’,N’-bis(benzyl-2-boronic acid)-bipyridinium dibromide (o-BBV) N N B B HO OH OH HO B OH HO B OH HO Br 2Br a b 48 49 50 Conditions: (a) NBS, AIBN, CCl4, reflux, 2 hrs, 50 %; (b) i. methanol, reflux; ii. 4,4’-dipyridyl, 63%. 46

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4.4.2 Studies on the binding of o-BBV to 3-acryloyl-D-glucose Having synthesized the boronic acid-modified qu encher, o-BBV, fluorescence studies were then carried out to investigate wh ether 3-acryloyl-D-glucose ( 19 ) can bind to o-BBV as shown in Scheme 4.3. Addition of 5 mg/mL of 3-acryloyl-D-glucose in pH 7.4 phosphate buffer to an aqueous solution of o-BBV (3X10-4 M) and pyranine (1X10-5 M) led to a 17% signal increas e in fluorescence intensity at max = 517 nm ( max = 511 nm has been reported in the literature), as plotted in Fig. 4.5. This shows that this quencher/dye combination is highly sensitive not only to glucose81 but also to 3-acryloyl-D-glucose ( 19 ). Scheme 4.5: Reversible complexation reaction of o-BBV/3-acryloyl-D-glucose. SO3 -O3S -O3S OH N N B B OH OH HO OH N N B B SO3 -O3S -O3S OH +4 H2O+ O HO O OH OH HO O OH O O O OH O HO O O O HO O O O pH 7.4 pyranine o-BBV Weak FluorescenceStrong Fluorescence 19 47

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Figure 4.5: Fluorescence spectra of pyranine (1X10-5 M, excitation = 461.8 nm, emission max = 517 nm) in 0.1 ionic strength aqueous phosphate buffer (pH 7.4) ( pyranine with o-BBV ( 1X10-4 M) ; pyranine with oBBV ( 1X10-4 M) and 3-acryloyl-D-glucose (5 mg/mL). 0 200000 400000 600000 800000 1000000 1200000 1400000 1600000472 479 486 493 50 0 507 514 521 528 535 542 5 49 55 6 563 570 577 584 591 598 605 61 2 619 626Wavelength (nm)Intensity 48

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4.4.3 Studies on the binding of o-BBV to glyconanoparticles Having established the sensing ability of pyranine/o-BBV complex towards 3-acryloyl-D-glucose ( 19 ), the same experiment was repeated for the glyconanoparticles ( GNP 19 and GNP 41 ) as shown in Scheme 4.4. Scheme 4.6: Binding of glyconanoparticles with o-BBV O HO O OH OH HO O pyranine o-BBV Weak Fluorescence Strong Fluorescence SO3 -O3S -O3S OH N N B B OH OH HO OH N N SO3 -O3S -O3S OH +4 H2O+ pH 7.4 O OH O OH O O B O O HO O HO O O B O prepared at 70 oC according to the formulation: butyl acrylate – 700 mg styrene – 300 mg sodium dodecyl sulfate – 30 mg potassium persulfate – 10 mg and water – 4 mL. In this case, a 27% enhancement of fluorescence intensity (Figure 4.6) was observed upon mixing a solution of GNP 19 with pyranine/o-BBV complex against a solution of nanoparticles* prepared with styrene/butyl acrylate. Prior to the experiments, the nanoparticle emulsions were centrifuged at 12000-13000 rpm (16100 X g) for 30 minutes to remove any solid polymer materials formed during the emulsion polymerization, and then dialyzed (membrane, 6000-8000 MWCO) overnight in de-ionized water in order to remove excess surfactants. o-BBV binding studies were performed by adding 1.00 mL of emulsion ( GNP 19 or GNP 41 ) to 4.00 mL of pyranine (1X10-5 M) /o-BBV(1X10-4 M) solution at pH 7.4. 49

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Figure 4.6: Fluorescence spectra of pyranine (8X10-6 M, excitation = 461.8 nm) in 0.1 ionic strength aqueous phosphate buffer (pH 7.4) ( pyranine with o-BBV (8X10-5 M) and emulsion* (20% by vol.) prepared with styrene/butyl acrylate; pyranine with o-BBV (8X10-5 M) and GNP 19 (20% by vol.; 5 mg/mL of 19 ). 0 200000 400000 600000 800000 1000000 1200000 1400000 1600000 1800000475 481 4 87 493 499 50 5 511 517 52 3 529 535 54 1 547 5 53 5 59 565 5 71 5 77 583 5 89 5 95 601 607Wavelength (nm)Intensity 50

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By changing the syst em from pyranine (1X10-5 )/ o-BBV (1X10-4 M) to pyranine (8X10-6 )/ o-BBV (8X10-5 M)/ emulsion* (20% by vol.) with a 20% dilution of the dye/quencher mixture, only 2% signal increase of the fluorescence intensity was observed. This increase could be due to the interaction of negatively charged polyacrylate nanoparticles to positively charged o-BBV lowering the quenching ability. Therefore, a 27% fluorescence enhancement from GNP 19 relative to the emulsion* (prepared with styrene/butyl acrylate) indicates the presence of a binding in teraction of a surface sugar with o-BBV Curiously, a relative fluorescence enhancement could not be observed upon using GNP 41 with the o-BBV/pyranine system. This may indicate that the highly lipophilic monomer 41 could allow for the entrapment of the carbohydrate moiety inside the nanoparticle during the polymerization, making it unavailable on the surface. As these studies revealed that the surface charge of the glyconanoparticle can affect the fluorescence intensity of dye/quencher systems, it may not be recommended that the boronic acid salts be used as reporter units in future quantification studies involving these anionically-charged nanoparticles. Instead, the watersoluble boronic acids, such as dibenzofuran-4-boronic acid (Figure 4.7) which exhibits unique fluorescence changes at three wavelengths upon binding with sugars in water83, may be more suitable for this purpose. Figure 4.7: Dibenzofuran-4-boronic acid O B HO OH In conclusion, polymeric glyconanoparticles with surface-exposed carbohydrates have been prepared by microemulsion polymerization w ith 3-acryloyl-D-glucose. Carbohydra te monomers with longer tether lengths, such as monomer 41 do not appear to be suitable for this preparation due to the higher lipophilicity. 51

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4.5 Conclusions and future work Throughout various fields of science and technology, a push towards the use of nano-scale technology is well underway. One area where nano-scale work is already prominent is within the field of drug delivery. The preparation of glyconanoparticle antibiotics, or glycona nobiotics, allows the Turos group to expand to other classes of antibiotics with poor lipophilicity. One of the key features of these glycosylated po lyacrylate nanoparticles is their binding ability with boronic acids, which may allow for novel applications in carbohydrate-ligand mediated targeted drug delivery. Also, this feature makes these glyconanoparticles ideal candidates for cancer therapy as ‘plug drugs’ (drugs which fit into the active sites) as researchers have sh own that a significant link exists between the presence of carbohydrates on the cancer cell surface an d their metastatic potential (capacity to spread around the body). Furthermore, functionalization of glyconanoparticles has been demonstrated for the first time, through the binding of the surface-exposed carbohydrates with water-soluble boronic acid salts. This may offer a straightforward, simple, synthetic pathway for the development of novel functionalized nanomaterials as shown in Figure 4.8. The designing of nanoparticles of different sizes could be useful for applications in cavitationmediated drug delivery for cancer treatments. 84 Efficacy and safety of cancer ch emo-and biotherapy are limited Figure 4.8: Post-functionalization of glyconanoparticles O OH OH O HO HO O O O HO HO O O B O X O O HO HO O O B O X Functionalization Derivatization by poor penetration of anti-cancer drugs from blood into tumor cells. Tumor blood vessel wall and cancer cell membrane create physiological barriers for anti-cancer drugs, in particular, promising macromolecular agents. The nanoparticles selectively delivered in tumor blood ve ssels can serve as nuclei for formation of cavitation microbubbles upon ultrasound irradiation. The interaction of the nanoparticles with the ultrasound results in cavitation (formation, growth, and collapse of microbubbles). The ultrasound induced cavitation is accompanied by strong local h ydrodynamic flows, which can produce th e rupture of tumor blood vessel wall and cancer cell membrane. This may enhance delivery of the drugs into cancer cells. Cavitation threshold, defined as minimal ultrasound pressure producing cavitation, is dependent on the size of the nanoparticle. Hence, the ability to synthesize glyconanoparticles in different sizes with different carbohydrate monomer contents may be useful to provide nano-polymeric tools for these applications. 52

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CHAPTER V MATERIALS AND METHODS 5.1 Synthetic Procedures All chemicals required for the synthesis of carbohydrate compounds were purchased from one of the following sources: Sigma Aldrich, Fisher Scientific, Across Organics, TCI Organic Chemicals, or Lancaster Research Chemicals. Most were used without further purification. Solvents were obtained from Fisher Scientific. Products were purified by flash chromatogr aphy using J. T. Baker chromatography silica gels (40 m). NMR spectra were recorded in either CDCl3 or D2O as indicated. 5.1.1 Preparation of O -isopropylidene protected carbohydrate acrylates 1,2:5,6-DiO -isopropylidine-D-glucofuranoside (6): Anhydrous D-glucose (10g) was stirred vigorously with 200 mL of acetone in an ice bath. Conc. H2SO4 (8 mL) was added in 1 mL portions at 10-15 min intervals, while maintaining the temperature at 5-10 oC. After the addition of the sulfuric acid, the vigorous stirring was continued for 5h, allowing the temperature to rise gradually to 20-25 oC. The solution was cooled again (ice bath), and 50% sodium hydroxide solution (12.25g of NaOH in 15 mL of water) was added with stirring to near neutrality. A small amount of sodium hydrogen carbonate was added to main tain the solution near neutrality. After standing overnight, the salts were removed by filtr ation, and the acetone solution was concentrated under reduced pressure to a thick syrup that solidifies on standing. The mixture was dissolved in chloroform in a water bath, and the solution was extracted with water to remove the monoO -isopropylidene derivative. The chloroform solution was concentrated under reduced pressure to provide a syrup and compound 5 was obtained after recrystallization from cyclohexane (11.8 g, 82%) as a white solid; mp 108-110 oC; 1H NMR (250MHz, CDCl3): 5.95 (d, 1H, J =3.6 Hz), 4.54 (d, 1H, J =3.6 Hz), 4.32 (m, 2H), 4.17 (m, 1H), 3.97 (m, 2H), 1.49 (s, 3H), 1.44 (s, 3H), 1.37 (s, 3H), 1.35 (s, 3H). 1,2:5,6-DiO -isopropylidine-D-glucofuranose-3-acrylate (1): To a stirred solution of 1,2:5,6-DiO isopropylidine-D-glucofuranoside ( 6 ) (2.0g, 7.69 mmol) and triethylamine (1.6 mL, 11.53 mmol) in CH2Cl2 (10 mL) acryloyl chloride (0.68 mL, 8.46 mmol) was added dropwise at 0 oC and and stirred at room temperature for 10 h. The reaction mixture was treated with water (25 mL) and extracted into CH2Cl2 (3X50 mL). The combined organic layers were washed with water (2X100 mL), dried (Na2SO4) and evaporated under reduced pressure. The residue was purified by column ch romatography (hexane-EtOAc, 8.5:1.5) to afford the compound (1.6 g, 68%) as a white solid; mp 66-68 oC; 1H NMR (250 MHz, CDCl3): 6.45 (d, 1H, J =17.1 Hz), 6.10 (dd, 1H, J =10.3, 17.2 Hz), 5.88 (m, 2H), 5.28 (m, 1H), 4.50 (d, 1H, J =3.7 Hz), 4.20 (m, 2H), 4.01 (m, 2H), 1.48 (s, 3H), 1.36 (s, 3H), 1.26 (s, 6H); 13C NMR (63 MHz, CDCl3): 164.6, 123.0, 127.6, 112.2, 109.2, 104.9, 83.1, 79.6, 76.1, 72.3, 67.0, 26.7, 26.6, 26.1, 25.1. 1,2:3,4-DiO -isopropylidine-D-galactopyranoside (7): 9g (0.05 mol) of finely powdered anhydrous Dgalactose, 20g (0.125 mol) of anhydrous CuSO4, 1 mL of conc. H2SO4 and 200 mL of anhydrous acetone were mixed together and was stirred 24 hrs. CuSO4 was removed by filtration and washed with anhydrous acetone. The washings and filtrate were neutralized by shaking with 9.4g (0.127 mol) of calcium hydroxide, and calcium sulfate was filtered off and washed w ith dry acetone. Solvents were removed under reduced pressure to afford the compound 7 ( 9.63 g, 74%) as a syrup.1H NMR (250MHz, CDCl3): 5.5 (d, 1H, J =5.0 Hz), 4.53 (dd, 1H, J =2.2, 7.9 Hz), 4.25(m, 2H), 3.83(m, 1H), 3.66(m, 2H), 2.92(m, 1H), 1.46(s, 3H), 1.38(s, 3H), 1.26(s, 6H). 53

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1,2:3,4-DiO -isopropylidine-D-galactopyranose-6-acrylate (2): To a stirred solution of 1,2:5,6-diO isopropylidine-D-galactopyranoside ( 7 ) (2.0g, 7.69 mmol) and triethylamine (1.6 mL, 11.53 mmol) in CH2Cl2 (10 mL) acryloyl chloride (0.68 mL, 8.46 mmol) was added dropwise at 0 oC and stirred at room temperature for 10 h. The reaction mixture was treated with water (25 mL) and extracted into CH2Cl2 (3X50 mL). The combined organic layers were washed with water (2X100 mL), dried (Na2SO4) and evaporated under reduced pressure to afford compound 2 (1.52 g, 65%) as a syrup. 1H NMR (250 MHz, CDCl3): 6.44 (d, 1H, J =17.1 Hz), 6.11 (dd, 1H, J =9.3, 17.0 Hz), 5.83 (d, 1H, J =9.7 Hz), 5.52 (s, 1H), 4.61 (m, 1H), 4.31 (m, 4H), 4.05 (m, 1H), 1.48 (s, 3H), 1.43 (s, 3H), 1.31 (s, 6H); 13C NMR (63 MHz, CDCl3): 166.0, 131.1, 128.1, 109.5, 108.7, 96.2, 70.9, 70.5, 70.3, 65.9, 63.4, 25.9, 24.9, 24.4. 2,3:5,6-DiO -isopropylidine-D-mannofuranoside (8): D-Mannose (10 g, 55.5 mmol) was suspended in 20 mL of anhydrous acetone to which was added in rapid succession fused ZnCl2 (12 g, 88.04 mmol) and a homogeneous mixture of P2O5 (2 g, 14.09 mmol) and 85%H3PO4 (4 g, 34.6 mmol). The mixture was stirred for 2 hrs until solution was complete and allowed to stand ov ernight at room temperature. The mixture was then made alkaline by adding an aqueous suspension of Na2CO3 and the precipitate of Zn2CO3 was filtered, washed with acetone, and the co mbined filtrate and washings were distilled in vacuo until most of the acetone was removed. The residue was purified by column chromatography (silica gel, hexane-EtOAc, 1:1) to afford the compound 8 (12.2 g, 85%) as a syrup.1H NMR (250 MHz, CDCl3): 5.38 (d, 1H, J =2.1 Hz), 4.82 (m, 1H), 4.63 (d, 1H, J =5.8 Hz), 4.37(m, 1H), 4.20(m, 1H), 3.04(m, 2H ), 1.45(s, 6H), 1.37(s, 3H), 1.32(s, 3H). 2,3:5,6-DiO -isopropylidine-D-mannofuranose-1-acrylate (3): To a stirred solution of 1,2:5,6-diO isopropylidine-D-mannofuranoside ( 8 ) (2.0g, 7.69 mmol) and triethylamine (1.6 mL, 11.53 mmol) in CH2Cl2 (10 mL) acryloyl chloride (0.68 mL, 8.46 mmol) was added dropwise at 0 oC and stirred at room temperature for 10 h. The reaction mixture was treated with water (25 mL) and extracted into CH2Cl2 (3X50 mL). The combined organic layers were washed with water (2X100 mL), dried (Na2SO4) and evaporated under reduced pressure. The residue was purified by column chromatography (hexane-EtOAc, 8.5:1.5) to afford the compound 3 (1.44 g 62%) as a syrup .1H NMR (250 MHz, CDCl3): 6.45 (d, 1H, J =18.3 Hz), 6.17 (s, 1H), 6.07 (dd, 1H, J =10.3, 17.1 Hz), 5.89 (d, 1H, J =10.4 Hz), 4.85 (m, 1H), 4.73 (m, 1H), 4.37 (m, 1H), 4.02 (m, 3H), 1.46 (s, 3H), 1.43 (s, 3H), 1.34 (s, 3H), 1.31 (s, 3H); 13C NMR (63 MHz, CDCl3): 164.3, 132.2, 127.7, 113.2, 109.3, 100.9, 85.0, 82.2, 79.2, 72.8, 66.7, 26.9, 25.8, 25.0, 24.5. Methyl 2,3-isopropylidine-D-ribofuranoside (9): A solution of dry D-ribose (5.0 g, 33.30 mmol) in 100 mL of acetone, 2,2-dimethoxypropane (10 mL, 68.5 mmol), and 20 mL of me thanol containing 2mL methanol saturated with hydrogen chloride at 0 oC was stirred at 25 overnight. The resulting solution was neutralized with pyridine and evaporated to a yellow oil. This oil was partitioned between 50 mL of water and 20 mL of ether. The water layer was extracted twice with 20 mL portions of ether, and the combined ether extracts were dried over MgSO4. Evaporation yielded a pale yellow oil which was purified by column chromatography (hexaneEtOAc,2:1 ) to afford compound 9 (4.7g, 86%) as an oil. 1H NMR (250 MHz, CDCl3): 4.91 (s, 1H), 4.78 (d, 1H, J =5.8 Hz), 4.54 (d, 1H, J =5.8 Hz), 4.35 (s, 1H), 3.58 (m, 2H), 3.37 (s, 3H), 3.17 (s, 1H), 1.42 (s, 3H), 1.26 (s, 3H). Methyl 2,3-isopropylidine-D-ribofuranose-5-acrylate (4): To a solution of methyl 2,3-isopropylidene-Dribofuranoside ( 9 ) (1.0 g, 4.9 mmol) in CH2Cl2 (5 mL) with triethylamine (1.0 mL, 7.3 mmol), acryloyl chloride (0.43 mL, 5.4 mmol) was added dropwise at 0 oC. The mixture was magnetically stirred for 30 min. at 0 oC and then for another 8 hrs at room temp erature. The reaction mixture was trea ted with water (15 mL) and extracted into CH2Cl2 (3 x 25 mL). The combined organic layers were washed with water (2 x 50 mL), dried over anhydrous MgSO4 and evaporated under reduced pressure. The re sidue was purified by column chromatography (silica gel, hexane-EtOAc, 8.5:1.5) to afford the compound 4 (1.04 g, 83%) as a syrup. 1H NMR (250 MHz, CDCl3): 6.42 (d, 1H, J =16.3 Hz, olefinic), 6.10 (dd, 1H, J =10.3, 17.3 Hz, olefinic), 5.82 (d, 1H, J =10.3 Hz, olefinic), 4.92 (s, 1H, H-1), 4.63 (d, 1H, J =5.9 Hz, H-3), 4.55 (d, 1H, J =5.9 Hz, H-2), 4.35 (t, 1H, J =6.9 Hz, H-4), 4.11 (m, 2H, H-5,5’), 3.25 (s, 3H, OCH3), 1.42 (s, 3H, CH3), 1.26 (s, 3H, CH3); 13C NMR (63 MHz, CDCl3): 165.5, 131.3, 127.9, 112.4, 109.2, 85.0, 84.0, 81.7, 64.5, 54.7, 26.3, 24.8. 54

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5.1.2 Preparation of O -acetal-protected acrylamide from glucosamine 1,3,4,6-TetraO -acetyl-D-glucosamine hydrochloride (12): To a solution of D-glucosamine hydrochloride (2.5 g, 11.6 mmol) in a freshly prepared aqueous solution of 1M NaOH (12 mL) under stirring was added p anisaldehyde (1.7 mL, 12.36 mmol). Then the mixture was refrigerated and after 2 h the precipitated product was filtered off and washed with cold wate r, and followed by a mixture of 1:1 EtOH-Et2O to give 2-deoxy-2-[ p methoxybenzylidene(amino)]-D-glucopyranose ( 10 ). This intermediate product (2.5g) was added successively to a cooled (ice-water) mixture of pyridine (13.5 mL) and Ac2O (7.5 mL). The mixture was stirred in ice bath ~ 1 h and then at room temperature overnight. The yellow solution was poured into 50 mL of ice-water. The precipitated white product was filtered, washed with cold water and dried. Subsequently this product, 1,3,4,6tetraO -acetyl-2-deoxy-2-[ p -methoxybenzylidene(amino)]-D-glucopyranose ( 11 ) (3.4 g),was dissolved in warm acetone (30 mL), and then HCl (5M, 1. 5 mL) was added with immediate form ation of a precipitate. The mixture was cooled, and after addition of Et2O (30 mL), it was stirred for 2 hrs and refrigerated overnight. The precipitated product was filtered, washed with Et2O and dried to give compound 12 (1.56 g, 56%) as a white solid; mp 234-236 oC; 1H NMR (250 MHz, D2O): 5.86 (m, 1H), 5.36 (m, 1H), 5.03 (m, 1H), 4.26 (m, 1H), 4.11 (m, 2H), 3.63 (m, 1H), 2.01 (m, 12H). N -Acryloyl 1,3,4,6-tetraO -acetyl-D-glucosamine (5): To a suspension of 1,3,4,6-tetraO -acetyl-Dglucosamine hydrochloride ( 12 ) (1.0g, 2.6 mmol) and triethylamine (0.54 mL, 3.9 mmol) in CH2Cl2 (10 mL) acryloyl chloride (0.23 mL, 2.86 mmol) was added dropwise at 0 oC and stired at room temperature for 10 h. The reaction mixture was treated with water (25 mL) and extracted into CH2Cl2 (3X50 mL). The combined organic layers washed with water (2X50 mL), dried (Na2SO4) and evaporated under reduced pressure. The residue was purified by column chromatography (hexane-EtOAc, 2:1) to afford the compound (0.67 g, 65%) as a white solid; mp 180 oC; 1H NMR (250 MHz, CDCl3): 6.28 (d, 1H, J =16.3 Hz), 5.99 (dd, 1H, J =10.8, 16.6 Hz), 5.70 (m, 3H), 5.18 (m, 2H), 4.42 (m, 1H), 4.31 (m, 1H), 4.15 (m, 1H), 3.81 (m, 1H) 2.09 (s, 3H), 2.04 (s, 3H), 2.01 (s, 3H), 1.6 (s, 3H); 13C NMR (63 MHz, CDCl3): 171.4, 170.7, 169.6, 169.3, 165.4, 130.1, 127.6, 92.6, 72.9, 72.6, 67.8, 61.7, 52.8, 20.9, 20.7, 20.6. 5.1.3 Preparation of carbohydrate acrylate monomers with bulky aryl protecting groups Methyl 2,3,5-triO -benzyl-D-ribofuranoside (15): A solution of D-ribose (2.5g, 16.65 mmol) in 50 mL of anhydrous methanol was cooled to 0 oC and treated with 0.25 mL of conc. H2SO4. After storage in the freezer overnight, the solution was neutralized by adding Na2CO3 then filtered, and the product, methyl -Dribofuranoside, was isolated by evaporating the solvent. This intermediate product (2.6g, 15.8 mmol) was dissolved in 25 mL of dry DMF and stirred with NaH (4.4g, 110.6 mmol, 60% dispersion in mineral oil ) for 2 h at room temperature. The mixture was cooled to 0 oC and benzyl bromide (18.1 mL, 79 mmol) was added dropwise to the mixture and stirring was continued overnight at room temperature. The mixture was cooled to 0 oC and excess NaH was decomposed by dropwise addition of MeOH (5 mL). The solution was concentrated in vacuo and diluted with EtOAc (125 mL) and water ( 65 mL). The organic phase was separated, washed with water (3X 30 mL) and brine (30 mL). The solvents we re evaporated and the resi due was purified by column chromatography (hexane-EtOAc, 9:1) to give compound 15 (2.6 g, 62 %) as a colorless syrup; 1H NMR (250 MHz, CDCl3): 7.33 (m, 15H), 4.95 (s, 1H), 4.59 (m, 6H), 4.34 (m, 1H), 4.07 (m, 1H), 3.88 (m, 1H), 3.61 (m, 2H), 3.34 (s, 3H). 2,3,5-TriO -benzyl-D-ribofuranose (16): Methyl 2,3,5-triO -benzyl-D-ribofuranoside ( 15 ) (2.5g, 9.9 mmol) was dissolved in 50 mL of dioxane and 12.5 mL of 0.1 N HCl was added and boiled gently till 12.5 mL of distillate was collected. After cooling, it was made up to its original volume by adding 12.5 mL of a mixture of 12 parts of water and 88 parts of dioxane (v./v.). More 0.1N HCl (12.5) mL was added and the process was repeated and 10 mL of the distillate was removed. The remaining solution was neutralized with 1 N NaOH, the solution was concentrated in vacuo The residual syrup was dissolved in CH2Cl2, washed with water (3X20 mL) and dried over anhydrous MgSO4. The crude product was purified by column chromatography to give the compound 16 (1.91g, 81 %) as a syrup; 1H NMR (250 MHz, CDCl3): 7.28 (m, 15H), 5.35 (s, 1H), 4.58 (m, br, 7H), 3.99 (m, 2H), 3.48 (m, 2H). 55

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2,3,5-TriO -benzyl-D-ribofuranose-1-acrylate(13): To a solution of 2,3,5-triO -benzyl-D-ribofuranose ( 16 ) (1.0 g, 4.19 mmol) in CH2Cl2 (5 mL) with triethylamine ( 0.87 mL, 6.28mmol), acryloyl chloride (0.37 mL, 4.6 mmol) was added dropwise at 0 oC. The mixture was magnetically stirred for 30 min. at 0 oC and then for another 8 hrs at room temperature. The reaction mixture was treated with water (15 mL) and extracted into CH2Cl2 (3 x 25 mL). The combined organic layers were washed with water (2 x 50 mL), dried over anhydrous MgSO4 and evaporated under reduced pressure. The residue was purified by column ch romatography (silica gel, hexane-EtOAc, 3:1) to afford the compound 13 (1.04 g, 83%) as a syrup. 1H NMR (250 MHz, CDCl3): 7.35 (m, 15H), 6.31 (m, 2H), 6.02 (m, 1H), 5.81 (m, 1H), 4.84 (m, 1H), 4.69 (m, br, 6H), 4.42 (m, 1H), 4.01 (m, 1H), 3.65 (m, 2H); 13C NMR (63 MHz, CDCl3): 164.6, 138.2, 137.5, 137.3, 131.8, 128.4, 128.2, 128.1, 127.9, 127.8, 127.5, 99.3, 81.6, 78.7, 77.6, 77.0, 76.8, 76.5, 73.2, 72.4, 72.0, 69.5. N -Acryloyl-1,3,4,6-tetraO -benzoyl-D-glucosamine (14): 2-Deoxy-2-[p-methoxybenzylidene (amino)]-Dglucopyranose ( 10 ) ( 0.5 g, 1.68 mmol) was added successively to a cooled (ice-water) mixture of pyridine (10 mL) and stirred. Benzoyl chloride (3.2 mL, 27.77 mmol) was added dropwise and stirred at room temperature for 5h. The mixture was washed with saturated NaHCO3 and extracted with Et2O, and washed with water. Solvents were evaporated in vacuo and the residue was dissolved in aceton e (30 mL), and then HCl (5M, 2 mL) was added with immediate formation of a precipitate. The mixture was cooled, and after addition of Et2O (30 mL), was stirred for 2 hrs at 0 oC. The precipitated product wa s filtered, washed with Et2O and dried to give 1,3,4,6-tetraO -benzoyl-D-glucosamine hydrochloride ( 18 ).To a suspension of compound 18 (0.5g, 0.79 mmol) and triethylamine (0.16 mL, 1.18mmol) in CH2Cl2 (10 mL) acryloyl chloride (0.07 mL, 2.86 0.87mmol) was added dropwise at 0 oC and stired at room temperature for 10 h. The reaction mixture was treated with water (25 mL) and extracted into CH2Cl2 (3X50 mL). The combined organic layers washed with water (2X50 mL), dried (Na2SO4) and evaporated under reduced pressure The residue was purified by column chromatography (hexane-EtOAc, 2:1) to afford compound 14 (0.44 g, 40%) as a syrup.1H NMR (250 MHz, CDCl3): 7.95 (m, 8H), 7.36 (m, 12H), 5.90 (m, 5H), 5.46 (m, 1H), 4.60 (m, 1H), 4.51 (m, 2H), 4.28 (m, 1H). 5.1.4 Deacetonation of carbohydrate acrylates 3-Acryloyl-D-glucose (19): 1,2:5,6-DiO -isopropylidine-D-glucofuranose-3-acrylate ( 1 ) (0.5 g, 1.59 mmol) was dissolved in 1 mL of 90% trifluoroacetic acid, stirred 30 min. at room temperature, and the solvents were removed in vacuo to afford compound 19 (0.33 g, 90%) as a syrup. 1H NMR (400MHz, D2O): 6.30 (dd, 1H, J 2.4, 17.2 Hz), 6.08 (dd, 1H, J =10.8,17.6 Hz), 5.84(dd, 1H, J =3.2,10.4 Hz), 5.04(m, 1H), 4.87(m, 1H), 3.773.26(m, 5H);13C NMR (63 MHz, D2O): 168.2, 168.0, 133.0, 132.8, 127.3, 127.2, 95.7, 91.9, 77.5, 75.6, 75.4, 72.3, 71.1, 69.7, 67.7, 60.4, 60.3. Methyl -D-ribofuranose-5-acrylate (20): Methyl 2,3O -isopropylidene-D-ribofuranose-5-acrylate ( 5 ) (0.50 g, 1.9 mmol) was dissolved in 80% acetic acid and stirred at 80 oC for 2 h. The solvent was then removed in vacuo and the residue was purified by column chromatography (silica gel, hexane-EtOAc, 1:6) to give compound 20 (0.28 g, 68%) as a syrup. 1H NMR (400 MHz, CDCl3): 6.41 (d, 1H, J =17.6 Hz, olefinic), 6.12 (dd, 1H, J =10.4,17.2 Hz, olefinic), 5.83 (d, 1H, J =10.8 Hz, olefinic), 4.79 (s, 1H, H-1), 4.40 (m, 1H), 4.15 (m, 3H), 3.97 (d, 1H, J =4.4 Hz), 3.59 (s, br, 2H, OH), 3.28 (s, 3H, OCH3);13C NMR (100 MHz, CDCl3): 166.1, 131.8, 128.2, 108.3, 80.9, 75.1, 72.1, 65.3, 55.2. 5.1.5 Preparation of nucleoside acrylates 2’, 3’O -Isoprpylidene cylidine (21): Cytidine (1 g, 4.11 mmol) and p-toluenesulfonic acid monohydrate (7.85 g, 41.26 mmol) were suspended in 150 ml of acetone and stirred for 2 hrs at room temperature. The precipitate formed was separated by filtration. This was dissolved in aq. NaHCO3 (1.2 eq) and stirred for an hour. The solvents were evaporated an d the residue was extracted with CHCl3. Compound 21 (0.83 g, 72%) was obtained after removing solvents as a syrup. 1H NMR (250 MHz, CDCl3): 8.95 (s, 2H), 7.79 (d, 1H, J =7.4 Hz), 7.48 (d, 1H, J =7.4 Hz), 5.55 (d, 1H, J =2.5 Hz), 5.25 (dd, 1H, J =2.6, 6.5 Hz), 5.09 (dd, 1H, J =3.1, 6.5 Hz), 4.41 (d, 1H, J =2.5, Hz), 3.95 (m, 1H), 3.88 (m, 1H), 3.86 (m, 1H), 3.85 (m, 1H), 3.60 (m, 1H), 1.61 (s, 3H), 1.38 (s, 3H). 56

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4N -Acetyl-2’,3’O -isopropylidene cytidine (22): 2’, 3’O -Isoprpylidene cylidine ( 21 ) (0.5g, 1.76 mmol ) was dissolved in 50 mL of anhydrous methanol and 0.5 mL of Ac2O was added and heated to reflux. Three portions of 0.5 mL Ac2O were added to the refluxing mixture at every 45 minutes and refluxed another hour. The solvents were evaporated under reduced pressure and the residue was washed with ether. The residue was purified by column chromatography to afford compound 22 (0.46g, 81 %) as a white solid; mp 115-117 oC; 1H NMR (250 MHz, CDCl3): 7.34 (d, 1H, J =7.4), 5.78 (d, 1H, J =7.2), 5.41 (s, 1H), 5.03 (m, 1H), 4.90 (m, 1H), 4.19 (d, 1H, J =2.5), 3.82 (m, 2H), 2.30 (s, 3H), 1.46 (s, 3H), 1.25 (s, 3H). 4N -Acetyl-5’O -acryloyl-2’,3’O -isopropylidene cytidine (23): To a stirred solution of 4N -acetyl-5’O acryloyl-2’,3’O -isopropylidene cytidine ( 22 ) (0.25g, 0.77 mmol) and triethylamine (0.16 mL, 1.15mmol) in CH2Cl2 (5 mL) acryloyl chloride (0.068 mL, 0.85mmol) was added dropwise at 0 oC and stirred at room temperature for 10 h. The reaction mixture was treated with water (15 mL) and extracted into CH2Cl2 (3X25 mL). The combined organic layers were washed with water (2X50 mL), dried (Na2SO4) and evaporated under reduced pressure. The residue was purified by column chromatography to afford compound 23 (0.17 g, 60%) as a white solid; mp 109 oC; 1H NMR (400MHz, CDCl3): 7.71(d, 1H, J =7.6 Hz), 7.39(d, 1H, J =7.2 Hz), 6.40(d, 1H, J =17.2 Hz), 6.02(dd, 1H, J =10.4, 17.2 Hz), 5.84(d, 1H, J =10.8 Hz), 5.68(s, 1H), 5.01(d, 1H, J =6.0 Hz), 4.84(m, 1H), 4.43(m, 3H), 2.93(s, 3H), 1.55(s, 3H), 1.33(s, 3H);13C NMR (63 MHz, CDCl3): 165.5, 163.2, 154.6, 146.3, 131.8, 127.6, 114.2, 96.8, 96.5, 86.4, 85.2, 81.3, 64.4, 27.1, 25.2, 24.9. 5.1.6 Preparation of carbohydrate-based acrylated acyl chlorides 3O -Acryloyl-1,2O -isopropylidene-D-glucofuranoside(29): 1,2:5,6-DiO -isopropylidine-D-glucofurano-se-3-acrylate ( 1 ) (0.5g, 1.59 mmol) was dissolved in 4 mL of 80% acetic acid and stirred 4hrs at room 80 oC. The solvents were removed in vacuo and the residue was purified by co lumn chromatography (silica gel, hexane-EtOAc, 1:1) to afford compound 29 (0.32 g, 62%) as a syrup. 1H NMR (400 MHz, D2O): 6.32 (d, 1H, J =20.0 Hz), 6.02 (m, 1H), 5.86 (m, 2H), 5.16 (m, 1H), 4.12 (m, 1H), 3.73 (m, 1H), 3.59 (m, 1H), 3.45 (m,1H), 1.36 (s, 3H), 1.17(s, 3H);13C NMR (63 MHz, D2O): 166.8, 133.3, 126.8, 113.0, 104.7, 82.2, 78.3, 75.9, 67.9, 63.1, 25.3, 25.0. 3O -Acryloyl-1,2O -isopropylidene-5,6 bis(carboxypropanoyl)-D-glucofuranose (30): 3O -Acryloyl-1,2O -isopropylidene-D-glucofuranoside ( 29 ) (0.3g, 1.09 mmol) and succinic anhydride (0.65g, 6.54 mmol) were stirred with Et3N (0.76 mL, 5.45 mmol) in CH2Cl2 (10 mL) at rt for 5h. The r eaction mixture was washed with 5% KHSO4, then water. The organic layer was dried over Na2SO4, filtered and evaporated to dryness. The brown oil obtained was chromatographed on silica gel (EtOAc) and the solid obtained was centrifuged in CH2Cl2. After evaporation of the solvent, compound 30 (0.34g, 65%) was obtained as a syrup. 1H NMR (400 MHz, CDCl3): 9.18 (br, s, 2H), 6.34 (m, 1H), 6.01 (m, 1H), 5.81 (m, 2H), 5.27 (m, 2H), 4.57(m, 3H), 4.13(m, 1H), 2.57(m, 8H), 1.46(s, 3H), 1.26(s, 3H);13C NMR (400 MHz, CDCl3): 177.8, 177.6, 172.0, 171.0, 164.9, 132.8, 127.3, 112.8, 105.3, 83.3, 76.8, 75.3, 68.1, 63.6, 29.0, 28.9, 28.8, 26.8, 26.4. 3O -Acryloyl-1,2O -isopropylidene-5,6 bis((chlorosuccinyl)oxy)-D-glucofuranose (27): Compound 30 (0.3g, 0.80 mmol ) was dissolved in 3 mL of thionyl chloride, stirred 45 min. at room temperature, and the solvents were removed in vacuo to afford compound 27 (0.31g, 98%) as a syrup. 1H NMR (400 MHz, CDCl3): 6.39 (m, 1H), 6.04 (m, 1H), 5.87 (m, 2H), 5.28 (m, 2H), 4.50 (m, 3H), 4.17 (m, 1H), 3.13 (m, 4H), 2.62 (m, 4H) 1.48 (s, 3H), 1.27 (s, 3H);13C NMR (100 MHz, CDCl3): 173.2, 172.9, 171.0, 170.1, 169.8, 165.7, 164.7, 132.5, 127.5, 112.8, 105.3, 83.3, 76.6, 75.2, 68.5, 63.8, 41.8, 29.3, 28.6, 26.9. 57

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6O -Acetyl-3O -acryloyl-1,2O -isopropylidene-D-glucofuranoside(31): 3O -Acryloyl-1,2O -isopropylid-ene-D-glucofuranoside ( 29 ) (0.4 g, 1.52 mmol) was dissolved in 10 mL of CH2Cl2 and cooled to -40 oC. Collidine (0.36 g, 3.04 mmol) was added to the solution and acetyl chloride was added and stirred at -40 oC for 3 h and at rt for 1 h. The mixture was washed with water and extracted into CH2Cl2 (3X25 mL). The solvents were evaporated and the residue was purified by column chromatogra phy to afford compound 31 (0.33 g, 72 %) as a syrup. 1H NMR (400 MHz, CDCl3): 6.70 (s, 1H), 6.42 (d, 1H, J =17.2 Hz), 6.06 (dd, 1H, J =10.4, 17.2 Hz), 5.84 (m, 2H), 5.32 (m, 2H), 4.52 (m, 1H), 4.36 (m, 1H), 4.20 (m, 1H), 4.09 (m, 1H), 3.85 (m, 1H), 2.00 (s, 3H), 1.45 (s, 3H), 1.25 (s, 3H);13C NMR (100 MHz, CDCl3): 171.4, 165.8, 157.3, 147. 8, 132.9, 127.4, 121.4, 112.5, 105.1, 79.0, 76.6, 66.6, 24.1, 20.9. 6O -Acetyl-3O -acryloyl-1,2O -isopropylidene-5-carboxypropanoyl-D-glucofuranose (32): 6O -Acetyl3O -acryloyl-1,2O -isopropylidene-D-glucofuranoside ( 31 ) (0.3 g, 0.98 mmol) and succinic anhydride (0.29 g, 2.96 mmol) were stirred with Et3N (0.34 mL, 2.45mmol) in CH2Cl2 (10 mL) at rt for 5h. The reaction mixture was washed with 5% KHSO4, then water. The organic layer was dried over Na2SO4, filtered and evaporated to dryness. The brown oil obtained was chromatographed on silica gel (EtOAc) and the solid obtained was centrifuged in CH2Cl2. After evaporation of the solvent, compound 32 (0.26 g, 65%) was obtained as a syrup. 1H NMR (400 MHz, CDCl3): 9.17 (br, s, 1H), 6.37 (d, 1H, J =17.2 Hz), 6.06 (dd, 1H, J =10.4, 17.2 Hz), 5.88(m, 2H), 5.34(d, 1H, J =2.8 Hz), 5.23(m, 1H), 4.51(m, 3H), 4.12(dd, 1H, J =5.6, 12.4 Hz), 2.55(m, 4H), 2.02(s, 3H), 1.51(s, 3H), 1.30(s, 3H);13C NMR (100 MHz, CDCl3): 177.3, 171.3, 171.2, 165.1, 132.9, 127.6, 113.0, 105.5, 83.6, 77.2, 77.1, 75.6, 68.4, 63.7, 29.3, 27.2, 26.7, 21.1. 6O -Acetyl-3O -acryloyl-1,2O -isopropylidene-5-(chlorosuccinyl)oxy-D-glucofuranose (28): 0.3 g of 32 was dissolved in 3 mL of thionyl chloride, stirred 45 min. at room temperature, and the solvents were removed in vacuo to afford compound 28 (0.34 g, 98%) as a syrup. 1H NMR (400 MHz, CDCl3): 6.35 (d, 1H, J =17.2 Hz), 6.04 (dd, 1H, J =10.4, 17.2 Hz), 5.87 (m, 2H), 5.33 (d, 1H, J =2.8 Hz), 5.17 (m, 1H), 4.52 (m, 3H), 4.08 (dd, 1H, J =5.6, 12.4 Hz), 3.10 (m, 3H), 2.57 (m, 3H), 2.00 (s, 3H), 1.49 (s, 3H), 1.27 (s, 3H);13C NMR (100 MHz, CDCl3): 172.8, 170.8, 169.8, 164.8, 132. 7, 127.3, 112.7, 105.3, 83.3, 76.8, 75.2, 68.6, 63.2, 41.7, 29.4, 28.6, 26.3, 20.8. 5.1.7 Preparation of antibiotic-conjugated carbohydrate monomers Monomer 34: To a stirred solution of N-sec -butylthio -lactam derivative 33 (0.2 g, 0.7 mmol) and triethylamine (0.29 mL, 2.1 mmol) in CH2Cl2 (5 mL) diacyl chloride 27 (0.173g, 0.42 mmol in 0.5 mL of CH2Cl2) was added dropwise at 0 oC and stirred at room temperature for 10 h. The reaction mixture was treated with water (10 mL) and extracted into CH2Cl2 (3X15 mL). The combined organic layers were washed with water (2X50 mL), dried (MgSO4) and evaporated under reduced pressure. The residue was purified by column chromatography (hexane-EtOAc, 2:1) to afford the compound 34 (0.22 g, 62%) as a syrup. 1H NMR (400 MHz, CDCl3): 7.35 (m, 8H), 6.35 (d, 1H, J =17.2 Hz), 6.14 (m, 2H), 6.03 (dd, 1H, J =10.8, 17.6 Hz), 5.84 (m, 2H), 5.47 (m, 2H), 5.25 (s, 1H), 5.09 (m, 1H), 4.45 (m, 2H), 4.33 (m, 1H), 4.05 (m, 1H), 3.04 (m, 2H), 2.17 (m, 8H), 1.56 (m, 4H), 1.47 (s, 3H), 1.26 (m, 9H), 0.96 (m, 6H);13C NMR (100 MHz, CDCl3): 171.2, 170.3, 169.8, 169.0, 164.7, 134.6, 123.6, 130. 7, 130.1, 130.0, 128.9, 127.3, 126.9, 112.7, 105.3, 83.3, 75.3, 68.0, 63.7, 63.6, 48.6, 28.5, 28.3, 26.9, 26.4, 19.2, 18.9, 11.3. Monomer 36: To a stirred solution of ciprofloxacin hydrochloride ( 35 ) (0.2g, 0.6 mmol) and triethylamine (0.5 mL, 3.6 mmol) in CH2Cl2 (8 mL) diacyl chloride 27 (0.148 g, 0.36 mmol in 0.5 mL of CH2Cl2) was added dropwise at 0 oC and stirred at room temperature for 10 h. The reaction mixture was treated with water (10 mL) and extracted into CH2Cl2 (3X15 mL). The combined organic layers were washed with water (2X50 mL), dried (MgSO4) and evaporated under reduced pressure. The re sidue was purified by column chromatography (EtOAc) to afford compound 36 (0.16 g, 50%) as a yellow solid; mp > 80 oC (decomposition);1H NMR (250 MHz, CDCl3): 14.87(br, s, 2H), 8.55(s, 2H), 7.78 (d, 2H, J =12.8 Hz), 7.32 (d, 2H, J =6.8 Hz), 6.43 (d, 1H, J =17.2 Hz), 6.14 (dd, 1H, J =10.3, 16.9 Hz), 5.91 (m, 2H), 5.35 (m, 2H), 4.54 (m, 3H), 4.21(m, 1H), 3.81(s, 4H), 3.74(s, 4H), 3.57 (s, 2H), 3.39(s, 4H), 3.30 (s, 4H), 2.70(s, 4H), 2.62(s, 4H), 1.40(s, 3H), 1.38(m, 4H), 1.30(s, 3H), 1.20(m, 4H). 58

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Monomer 37: To a stirred solution of ciprofloxacin hydrochloride ( 35 ) (0.2g, 0.6 mmol) and triethylamine (0.5 mL, 3.6 mmol) in CH2Cl2 (8 mL) acyl chloride 28 (0.31g, 0.66 mmol in 0.5 mL of CH2Cl2) was added dropwise at 0 oC and stirred at room temperature for 10 h. The reaction mixture was treated with water (10 mL) and extracted into CH2Cl2 (3X15 mL). The combined organic layers were washed with water (2X50 mL), dried (MgSO4) and evaporated under reduced pressure. The re sidue was purified by column chromatography (EtOAc) to afford compound 37 (0.23 g, 56%) as a syrup. 1H NMR (400 MHz, CDCl3): 8.64(s, 1H), 7.90 (d, 1H, J =12.4 Hz), 7.32 (d, 1H, J =6.8 Hz), 6.39 (d, 1H, J =17.2 Hz), 6.05 (dd, 1H, J =10.4, 17.2 Hz), 5.88 (d, 1H, J =3.6 Hz), 5.86 (d, 1H, J =10.4 Hz), 5.32 (d, 1H, J =2.8 Hz), 5.24(m, 1H), 4.52(m, 3H), 4.12 (dd, 1H, J =5.2, 12.0 Hz), 3.79(m, 2H), 3.68(m, 2H), 3.53(m, 1H), 3.33(m, 2H), 3.25(m, 2H), 2.59(m, 4H), 2.02(s, 3H), 1.49(s, 3H), 1.37(m, 2H), 1.27(s, 3H), 1.18(m, 2H);13C NMR (100 MHz, CDCl3): 177.1, 171.8, 170.8, 169.6, 166.9, 164.8, 155.0, 152.5, 147.6, 145.6, 145.5, 139.1, 132.3, 127.7, 120.2, 112.7, 112.4, 108.2, 105.3, 105.2, 83.3, 75.5, 68.0, 63.3, 53.6, 50.2, 49.5, 45.2, 41 .5, 35.5, 29.4, 28.1, 26.9, 26.4, 20.9, 8.4. Monomer 40: 6-Aminopenicillanic acid (2 g, 0.924 mmol) and N O -bis(trimethylsilyl) acetamide ( 0.21 g, 1.02 mmol) were added to CH2Cl2 (10 mL) and stirred at rt overnight. Triethylamine (0.19 mL, 1.38 mmol) was added and cooled on ice. Acyl chloride 28 (0.52 g, 1.11 mmol in 0.5 mL of CH2Cl2) was added dropwise and stirred at room temperature for 10 h. The reactio n mixture was treated with water (10 mL) and extracted into CH2Cl2 (3X15 mL). The combined organic layers were washed with water (2X50 mL), dried (MgSO4) and evaporated under reduced pressure. The residue was pu rified by column chromat ography (EtOAc) to afford compound 40 (0.23 g, 56%) as a syrup. 1H NMR (400 MHz, CDCl3): 6.55 (d, 1H, J =8.8 Hz), 6.39 (d, 1H, J =17.2 Hz), 6.02 (m, 1H), 5.88 (m, 2H), 5.63 (dd, 1H, J =4.0, 8.8 Hz), 5.48 (d, 1H, J =4.0 Hz), 5.31 (m, 1H), 5.19 (m, 1H), 4.48 (m, 3H), 4.04 (m, 1H), 2.55 (m, 4H), 2.01 (s, 3H), 1.62 (s, 3H), 1.54 (s, 3H), 1.48 (s, 3H), 1.27 (s, 3H). 5.1.8 Preparation of long-cha in acrylated glucose monomers 11-Acryloyloxy undecan-1-ol (44): Sodium hydroxide (13.8 g, 0.345 mol) dissolved in deionized water was placed in a round bottomed flask and ke pt in an ice bath for 10 minutes. Ac rylic acid (23.7 mL 0.345 mol) was added dropwise under stirring to the above solution. After 30 minutes, the reaction mixture was freeze-dried. The product was then dissolved in methanol and precipita ted with diethyl ether. After filtering, sodium acrylate ( 43 ) (29.2 g, 0.31 mol) was obtained in 90% yield. Sodi um acrylate ( 1.65 g, 17.5 mmol), 11-bro moundecan-1ol (1.0 g, 4.0 mmol), n-tetrabutylammonium bromide ( 0.375 g, 1.08 mmol), 2,6,-di-t-butyl-4-methyl phenol ( 2 mg, 0.009 mmol) dissolved in deionized ( 3.5 mL) and chloroform (2 mL) were placed in a round-bottomed flask. The reaction vessel was placed in an oil bath at 100 oC, and vigorous magnetic stirring was applied for three days. After that time, the chloroform layer wa s washed with 2% NaOH solution (4X25 mL) and distilled water (4X25 mL). The organic layer was dried over magnesium sulfate, and the solvent evaporated and compound 44 (0.58 g, 58%) was obtained as a yellowish oil. 1H NMR (250 MHz, CDCl3): 6.28 (dd, 1H, J =1.6, 17.2 Hz), 5.96 (dd, 1H, J =10.2, 17.2 Hz), 5.82 (dd, 1H, J =1.6, 10.2 Hz), 4.03 (t, 2H, J =6.8), 3.48 (t, 2H, J =6.6 Hz), 3.24 (m, 1H), 1.13 (br, s, 18H). Acrylated acid 45: Compound 44 (0.3 g, 1.24 mmol) and succinic anhydride (0.37 g, 3.74 mmol) were stirred with Et3N (0.35 mL, 2.49 mmol) in CH2Cl2 (5 mL) at rt for 5h. The reaction mixture was washed with 5% KHSO4, then water. The organic layer was dried over Na2SO4, filtered and evaporated to dryness. The residue was purified by column chromatography to afford compound 45 (0.33 g, 80%) as a syrup. 1H NMR (400 MHz, CDCl3): 6.37 (d, 1H, J =17.2 Hz), 6.08 (dd, 1H, J =10.4, 17.6 Hz), 5.78 (d, 1H, J =10.4 Hz), 4.08 (m, 4H), 2.59 (m, 4H), 1.59 (m, 4H), 1.23 (m, 14H);13C NMR (63 MHz, CDCl3): 177.9, 172.2, 166.4, 130.5, 128.6, 65.0, 64.7, 29.4, 29.2, 28.9, 28.5, 25.8. 59

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Acrylate 46: Acid 45 (0.31 g, 0.90 mmol) was dissolved in CH2Cl2 (5 mL) and EDCI (0.26 g, 1.36 mmol) was added and stirred for 10 minutes. Di acetone-D-glucose (0.24, 0.90 mmol ) and a catalytic amount of DMAP were added and stirred overnight at rt. The solvents were evaporated and the re sidue was purified by column chromatography (hexane-EtOAc, 1:1) to afford the compound 46 (0.39 g, 75%) as a syrup. 1H NMR (400 MHz, CDCl3): 6.36 (d, 1H, J =17.6 Hz), 6.07 (m, 1H), 5.81 (m, 2H), 5.20 (s, 1H), 4.45 (m, 1H), 4.04 (m, 8H), 2.60 (m, 4H), 1.59 (m, 4H), 1.46 (s, 3H), 1.35 (s, 3H), 1.25 (m, 20H);13C NMR (63 MHz, CDCl3): 172.1, 171.0, 130.4, 128.6, 112.2, 109.3, 105.0, 83.2, 79.7, 76.3, 72.4, 67.2, 65.0, 64.6, 29.4, 29.2, 29.1, 28.9, 28.6, 26.8, 26.7, 26.2, 25.8, 25.2. Monomer 41: Compound 46 (0.3g, 0.51 mmol) was dissolved in 1 mL of 90% trifluoroacetic acid, stirred 30 min. at room temperature, and the solvents were removed in vacuo The residue was purified by column chromatography to afford the compound 41 (0.33g, 90%) as a syrup. 1H NMR (400 MHz, CDCl3): 6.34 (d, 1H, J =17.6 Hz), 6.09 (dd, 1H, J =10.8, 17.6 Hz), 5.80 (d, 1H, J =10.4 Hz), 5.25 (m, 1H), 4.53 (m, 1H), 4.11 (m, 8H), 3.59 (m, 1H), 2.66 (m, 4H), 1.61 (m, 4H), 1.27 (m, 16H). 5.1.9 Preparation of the boronic acid salt o-Bromomethylphenylboronic acid (50): o-Tolylboronic acid (1.0 g, 7.36 mmol), N-bromosuccinimide (1.57 g, 8.84 mmol) and AIBN (0.16 g, 0.97 mmol) were dissolved in anhydrous carbon tetrachloride (100 mL) and the solution was refluxed for 2 h under a nitrogen atmos phere. After cooling, the precipitate was removed by filtration and the filtrate was washed with water (40 mL). The organic la yer was separated, dried over MgSO4 and concentrated in vacuo to dryness. The reprecipitation of the residual solid from chloroform to hexane gave o-bromomethylphenylboronic ( 50 ) acid (0.79 g, 50 %) as a white solid; mp 138-142 oC;1H NMR (63 MHz, CDCl3): 8.41-7.50 (m, 4H), 5.16 (s, 2H). 4’,4’-N’,N’-bis(Benzyl-2-boronic acid)-bipyridinium dibromide (o-BBV) (48): Compound 50 (0.5 g, 2.32 mmol), 4,4’-dipyridyl ( 0.18 g, 1.16 mmol) and anhydrous CH3OH ( 12 mL) were added to an oven-dried 20 mL test tube and heated allowing the solvents to evaporate slowly until a paste appeared. Acetone was added and the precipitate was removed by filtratio n. The precipitate was washed with acetone-H2O (24:1) twice to afford compound 48 (0.27 g, 63 %) as a light yellowish solid; mp >229 oC (decomposition); 1H NMR (250 MHz, CDCl3): 8.98 (d, 2H, J =6.9 Hz), 8.40 (d, 2H, J =6.8 Hz), 7.72 (m, 1H), 7.52 (m, 3H), 6.00 (s, 2H). 5.2 Glyconanoparticle characterization 5.2.1 SEM analysis SEM measurements of the glyconanop articles were captured on a Hitachi S800 SEM instrument in the USF Department of Engineering. Samples were prepared by placing a drop of diluted emulsion (1 L of emulsion in 30 mL of de-ionized distilled water) on the silicon wafer and evaporating the solvent by air blowing prior to coating with gold sputter under high vacuum. The gold-coated glyconanoparticles were then observed by SEM. 5.2.2 DLS analysis DLS analyses were performed using a UPA 150 Honeywell MicroTrac DLS instrument (University of Florida Particle Engineering Research Center) equipped with a laser beam at 780 nm. The sample was diluted with deionized distilled water (0.05 mL of emulsion in 9.95 mL of water, polymer concentration 0.1% wt) and sonicated for 3 minutes for the analysis. The value is expressed in weight-averaged scales as unimode at a scattering angle of 180 o (backscatter) at a temperature of 25 oC. 60

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5.3 Microbiological test procedures The following baceria were used for the antimicr obial evaluation of carbohydrated antibiotic drug monomers: Bacillus anthracis (Sterne strain), Staphylococcus aureus USF525 (ATCC 25923), Staphylococcus aureus USF652-658 (obtained from Lakeland Regional Medical Center) 5.3.1 MIC determinations Media preparation: The minimum inhibitory concentrations were determined by agar plate dilution. The test media were prepared in 24 well plates (Coster 3524, Cambridge, MA) by adding a known concentration of the test drug in DMSO together w ith a solution of Mueller-Hinton II agar (Becton-Dickinson Laboratories, Cockeysville, MD) for a total volume of 1 ml in each well. Calculations of the overall concentration of antibiotic in the well were standardized by measuring from a 1 mg/ml stock solution of the test drug. At this concentration, the microliter quantity is equivalent to the micrograms in solution. The amount of agar solution added to the wells was determined by subt racting 1000 L from the quan tity of test drug in each well to give a combined volume of 1 mL. Following preparation of the well plates, the media were allowed to solidify at room temperature for 24 hours before inoculation. Inoculation: From an 24 hour culture of each organism on tryp tic soy agar (TSA) plates (BectonDickinson Laboratories, Cockeysville, MD ), the staphylococcal strains were gr own overnight in 5 mL of tryptic soy broth (Difco Laboratories, Detroit, MI) at 37 oC. One microliter of each cultu re was then applied to the appropriate well of agar and incubated at 37 oC overnight. After 24 hr, the MIC were determined by examining the wells visually for growth. 5.4 Fluorescence experiments Fluorescence experiments were done using a PC1 photon counting spectrofluorometer at Larsen lab in the Department of Chemistry. 100 mL of dye/quencher solution was prepared in pH 7.4 aqueous phosphate buffer of 0.1 ionic strength. Pyranine and o-BBV concentrations were set to be 1X10-5 and 1X10-4 respectively. 25 mg of acrylate 19 was dissolved in 5.00 mL of dye/quencher solution for sensing studies of 19 1.00 mL of emulsion GNP 19 was mixed with 4.00 mL of dye/quencher solution for sensing studies of GNP 19 The excitation wavelength was 461.8 nm and the fluorescence emission spectrum was recorded between 470 and 630 nm. 61

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CHAPTER VI 1H and 13C NMR SPECTRA Spectrum 6.1: 1H NMR (250 MHz, CDCl3) ( 1 ) Spectrum 6.2: 13C NMR (63 MHz, CDCl3) ( 1 ) 62O O O O O O O

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Spectrum 6.3: 1H NMR (250 MHz, CDCl3) ( 2 ) Spectrum 6.4: 13C NMR (63 MHz, CDCl3) ( 2 ) 63 O O O O O O O

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Spectrum 6.5: 1H NMR (250 MHz, CDCl3) ( 3 ) Spectrum 6.6: 13C NMR (63 MHz, CDCl3) ( 3 ) 64 O O O O OO O

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Spectrum 6.7: 1H NMR (250 MHz, CDCl3) ( 4 ) Spectrum 6.8: 13C NMR (63 MHz, CDCl3) ( 4 ) 65 O O OO O O

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Spectrum 6.9: 1H NMR (250 MHz, CDCl3) ( 5 ) Spectrum 6.10: 13C NMR (63 MHz, CDCl3) ( 5 ) 66 O OAc HN AcO AcO AcO O

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Spectrum 6.11: 1H NMR (250 MHz, CDCl3) ( 13 ) Spectrum 6.12: 13C NMR (63 MHz, CDCl3) ( 13 ) 67 O OBn BnO BnO O O

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Spectrum 6.13: 1H NMR (250 MHz, CDCl3) ( 14 ) 68 O OBz N H O OBz BzO OBz

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Spectrum 6.14: 1H NMR (400 MHz, D2O) ( 19 ) Spectrum 6.15: 13C NMR (63 MHz, D2O) ( 19 ) 69 O OH OH O HO HO O

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Spectrum 6.16: 1H NMR (400 MHz, D2O) ( 20 ) Spectrum 6.17: 13C NMR (100 MHz, D2O) ( 20 ) 70 O OH HO O O O

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Spectrum 6.18: 1H NMR (400 MHz, CDCl3) ( 23 ) Spectrum 6.19: 13C NMR (63 MHz, CDCl3) ( 23 ) 71 O OO O N N O H N O O

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Spectrum 6.20: 1H NMR (400 MHz, CDCl3) ( 27 ) Spectrum 6.21: 13C NMR (100 MHz, CDCl3) ( 27 ) 72 O O O O O O O O Cl O O Cl O

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Spectrum 6.22: 1H NMR (400 MHz, CDCl3) ( 28 ) Spectrum 6.23: 13C NMR (100 MHz, CDCl3) ( 28 ) 73 O O O O O O O O O Cl O

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Spectrum 6.24: 1H NMR (400 MHz, CDCl3) ( 30 ) Spectrum 6.25: 13C NMR (100 MHz, CDCl3) ( 30 ) 74 O O O O O O O O OH O O HO O

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Spectrum 6.26: 1H NMR (400 MHz, CDCl3) ( 32 ) Spectrum 6.27: 13C NMR (100 MHz, CDCl3) ( 32 ) 75 O O O O O O O O O HO O

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Spectrum 6.28: 13C NMR (100 MHz, CDCl3) ( 34 ) 76 O O O O O O O O O O O O O N N O Cl S O S Cl

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Spectrum 6.29: 1H NMR (400 MHz, CDCl3) ( 36 ) 1.5725 2.0797 1.9855 2.1464 1.0000 3.2024 2.1619 3.1419 1.5678 17.801 8.9607 16.347 14.8719 8.5518 7.7858 7.7346 7.3279 7.3008 6.4394 6.3705 6.1413 6.1003 6.0737 6.0314 5.9266 5.9121 5.8634 5.3582 5.2724 4.6333 4.5859 4.5461 4.5335 4.5057 4.4685 4.2424 4.2196 4.1937 4.1704 3.8167 3.7479 3.5717 3.3936 3.3065 2.7059 2.6270 1.5288 1.4075 1.3842 1.3033 1.2642 1.2333 1.2036 1.1954 (ppm) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 cipro(bis) 77 O O O O O O O O O O O N N N O HOOC F N N N O COOH F

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Spectrum 6.30: 13C NMR (100 MHz, CDCl3) ( 37 ) 78 O O O O O O O O O O N N N O HOOC F

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Spectrum 6.31: 1H NMR (400 MHz, CDCl3) ( 40 ) 79 O O O O O O O O O O N S H N O HOOC

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Spectrum 6.32: 1H NMR (400 MHz, CDCl3) ( 41 ) 80 O O O O O O OH OH O HO HO

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Spectrum 6.33: 1H NMR (400 MHz, CDCl3) ( 45 ) Spectrum 6.34: 13C NMR (63 MHz, CDCl3) ( 45 ) 81 O O O O O OH

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Spectrum 6.35: 1H NMR (400 MHz, CDCl3) ( 46 ) Spectrum 6.36: 13C NMR (63 MHz, CDCl3) ( 46 ) 82 O O O O O O O OO O O

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Spectrum 6.37: 1H NMR (250 MHz, D2O) ( 48 ) 1.5887 1.7802 0.9807 2.8379 2.0000 Integral 8.9862 8.9585 8.4059 8.3787 7.7201 7.6961 7.6898 7.5407 7.5212 7.5110 7.4902 7.4845 7.4757 7.4586 7.4523 6.0036 (ppm) 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 83 N N B B HO OH OH HO 2Br

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ABOUT THE AUTHER Sampath Abeylath received B.Sc(special) degree in chemis try with second class (upp er division) honors from the University of Ruhuna in Sri Lanka. Having worked as an Asst. Lecturer in chemistry at the same university and a research officer at Industrial T echnology Institute in Sri Lanka, he pursued a doctorate in chemistry in the synthetic laboratory of Professor Edward Turos. Sampath continues to follow his research interest in carbohydrate chemistry, as we ll as medicinal chemistry.